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Sample records for proteomic study revealed

  1. Talaromyces marneffei Genomic, Transcriptomic, Proteomic and Metabolomic Studies Reveal Mechanisms for Environmental Adaptations and Virulence

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    Susanna K. P. Lau

    2017-06-01

    Full Text Available Talaromyces marneffei is a thermally dimorphic fungus causing systemic infections in patients positive for HIV or other immunocompromised statuses. Analysis of its ~28.9 Mb draft genome and additional transcriptomic, proteomic and metabolomic studies revealed mechanisms for environmental adaptations and virulence. Meiotic genes and genes for pheromone receptors, enzymes which process pheromones, and proteins involved in pheromone response pathway are present, indicating its possibility as a heterothallic fungus. Among the 14 Mp1p homologs, only Mp1p is a virulence factor binding a variety of host proteins, fatty acids and lipids. There are 23 polyketide synthase genes, one for melanin and two for mitorubrinic acid/mitorubrinol biosynthesis, which are virulence factors. Another polyketide synthase is for biogenesis of the diffusible red pigment, which consists of amino acid conjugates of monascorubin and rubropunctatin. Novel microRNA-like RNAs (milRNAs and processing proteins are present. The dicer protein, dcl-2, is required for biogenesis of two milRNAs, PM-milR-M1 and PM-milR-M2, which are more highly expressed in hyphal cells. Comparative transcriptomics showed that tandem repeat-containing genes were overexpressed in yeast phase, generating protein polymorphism among cells, evading host’s immunity. Comparative proteomics between yeast and hyphal cells revealed that glyceraldehyde-3-phosphate dehydrogenase, up-regulated in hyphal cells, is an adhesion factor for conidial attachment.

  2. Fish gut-liver immunity during homeostasis or inflammation revealed by integrative transcriptome and proteome studies

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    Wu, Nan; Song, Yu-Long; Wang, Bei; Zhang, Xiang-Yang; Zhang, Xu-Jie; Wang, Ya-Li; Cheng, Ying-Yin; Chen, Dan-Dan; Xia, Xiao-Qin; Lu, Yi-Shan; Zhang, Yong-An

    2016-11-01

    The gut-associated lymphoid tissue, connected with liver via bile and blood, constructs a local immune environment of both defense and tolerance. The gut-liver immunity has been well-studied in mammals, yet in fish remains largely unknown, even though enteritis as well as liver and gallbladder syndrome emerged as a limitation in aquaculture. In this study, we performed integrative bioinformatic analysis for both transcriptomic (gut and liver) and proteomic (intestinal mucus and bile) data, in both healthy and infected tilapias. We found more categories of immune transcripts in gut than liver, as well as more adaptive immune in gut meanwhile more innate in liver. Interestingly reduced differential immune transcripts between gut and liver upon inflammation were also revealed. In addition, more immune proteins in bile than intestinal mucus were identified. And bile probably providing immune effectors to intestinal mucus upon inflammation was deduced. Specifically, many key immune transcripts in gut or liver as well as key immune proteins in mucus or bile were demonstrated. Accordingly, we proposed a hypothesized profile of fish gut-liver immunity, during either homeostasis or inflammation. Current data suggested that fish gut and liver may collaborate immunologically while keep homeostasis using own strategies, including potential unique mechanisms.

  3. Transcriptome and Proteome Studies Reveal Candidate Attachment Genes during the Development of the Barnacle Amphibalanus Amphitrite

    KAUST Repository

    Al-Aqeel, Sarah; Ryu, Tae Woo; Zhang, Huoming; Chandramouli, Kondethimmanahalli; Ravasi, Timothy

    2016-01-01

    The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41%. We identified 65,784 expressed contigs, and a total of 1387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development. Proteomics data are available via ProteomeXchange with identifier PXD004679.

  4. Transcriptome and Proteome Studies Reveal Candidate Attachment Genes during the Development of the Barnacle Amphibalanus Amphitrite

    KAUST Repository

    Al-Aqeel, Sarah

    2016-09-21

    The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI, and cyprid) from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41%. We identified 65,784 expressed contigs, and a total of 1387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development. Proteomics data are available via ProteomeXchange with identifier PXD004679.

  5. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

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    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  6. Transcriptome and proteome studies reveal candidate attachment genes during the development of the barnacle Amphibalanus Amphitrite

    Directory of Open Access Journals (Sweden)

    Sarah Al-Aqeel

    2016-09-01

    Full Text Available The acorn barnacle, Balanus amphitrite, is the main biofouling organism in marine environments. In the present study we profiled the transcriptome and proteome of B. amphitrite at different life stages (nauplius II, nauplius VI and cyprid from the Red Sea, where the average water surface temperature is 34°C and the salinity reaches 41‰. We identified 65,784 expressed contigs, and a total of 1,387 expressed proteins measured by quantitative proteomics. We found that osmotic stress, salt stress, hyperosmotic response and the Wnt signaling pathway were strongly up-regulated during the planktonic stage, while the MAPK pathway, lipid metabolism, and cuticle development genes were down-regulated. In the transition stage between the nauplius VI and the cyprid, genes that are involved in blood coagulation, cuticle development and eggshell formation were highly up-regulated, while the nitric oxide pathway, which stimulates the swimming and feeding response in marine invertebrates, was down-regulated. We are able to report for the first time that sound sensory system proteins are highly abundant in the nauplius VI stage, implying that these proteins are good targets for the development of new antifouling compounds. The results presented here together with the new genome-wide datasets for a non-model specie represent an important resource for the study of biofouling and development.

  7. Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

    Science.gov (United States)

    Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

    2016-10-07

    Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

  8. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

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    Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

    2012-01-10

    The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  9. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

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    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  10. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

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    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  11. Proteomic properties reveal phyloecological clusters of Archaea.

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    Nela Nikolic

    Full Text Available In this study, we propose a novel way to describe the variety of environmental adaptations of Archaea. We have clustered 57 Archaea by using a non-redundant set of proteomic features, and verified that the clusters correspond to environmental adaptations to the archaeal habitats. The first cluster consists dominantly of hyperthermophiles and hyperthermoacidophilic aerobes. The second cluster joins together halophilic and extremely halophilic Archaea, while the third cluster contains mesophilic (mostly methanogenic Archaea together with thermoacidophiles. The non-redundant subset of proteomic features was found to consist of five features: the ratio of charged residues to uncharged, average protein size, normalized frequency of beta-sheet, normalized frequency of extended structure and number of hydrogen bond donors. We propose this clustering to be termed phyloecological clustering. This approach could give additional insights into relationships among archaeal species that may be hidden by sole phylogenetic analysis.

  12. Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility

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    Ashok Agarwal

    2016-01-01

    Full Text Available To study the major differences in the distribution of spermatozoa proteins in infertile men with varicocele by comparative proteomics and validation of their level of expression. The study-specific estimates for each varicocele outcome were combined to identify the proteins involved in varicocele-associated infertility in men irrespective of stage and laterality of their clinical varicocele. Expression levels of 5 key proteins (PKAR1A, AK7, CCT6B, HSPA2, and ODF2 involved in stress response and sperm function including molecular chaperones were validated by Western blotting. Ninety-nine proteins were differentially expressed in the varicocele group. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in the varicocele group. Key protein functions affected in the varicocele group were spermatogenesis, sperm motility, and mitochondrial dysfunction, which were further validated by Western blotting, corroborating the proteomics analysis. Varicocele is essentially a state of energy deprivation, hypoxia, and hyperthermia due to impaired blood supply, which is corroborated by down-regulation of lipid metabolism, mitochondrial electron transport chain, and Krebs cycle enzymes. To corroborate the proteomic analysis, expression of the 5 identified proteins of interest was validated by Western blotting. This study contributes toward establishing a biomarker "fingerprint" to assess sperm quality on the basis of molecular parameters.

  13. Proteomics reveals the effects of sustained weight loss on the human plasma proteome

    DEFF Research Database (Denmark)

    Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

    2016-01-01

    Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed...... by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly...

  14. DIGE proteome analysis reveals suitability of ischemic cardiac in vitro model for studying cellular response to acute ischemia and regeneration.

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    Sina Haas

    Full Text Available Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy.With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research.

  15. Characteristics of the tomato chromoplast revealed by proteomic analysis

    OpenAIRE

    Barsan, Cristina; Sanchez-Bel, Paloma; Rombaldi, César Valmor; Egea, Isabel; Rossignol, Michel; Kuntz, Marcel; Zouine, Mohamed; Latché, Alain; Bouzayen, Mondher; Pech, Jean-Claude

    2010-01-01

    Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism ...

  16. Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

    OpenAIRE

    Sun, Su; Xie, Shangxian; Cheng, Yanbing; Yu, Hongbo; Zhao, Honglu; Li, Muzi; Li, Xiaotong; Zhang, Xiaoyu; Yuan, Joshua S.; Dai, Susie Y.

    2017-01-01

    Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass,...

  17. When proteomics reveals unsuspected roles: the plastoglobule example

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    Claire eBréhélin

    2013-04-01

    Full Text Available Plastoglobules are globular compartments found in plastids. Before initial proteomic studies were published, these particles were often viewed as passive lipid droplets whose unique role was to store lipids coming from the thylakoid turn-over, or to accumulate carotenoids in the chromoplasts. Yet, two proteomic studies, published concomitantly, suggested for the first time that plastoglobules are more than "junk cupboards" for lipids. Indeed, both studies demonstrated that plastoglobules do not only include structural proteins belonging to the plastoglobulin / fibrillin family, but also contain active enzymes. The specific plastoglobule localization of these enzymes has been confirmed by different approaches such as immunogold localization and GFP protein fusions, thus providing evidence that plastoglobules actively participate in diverse pathways of plastid metabolism. These proteomic studies have been the basis for numerous recent works investigating plastoglobule function. However, a lot still needs to be discovered about the molecular composition and the role of plastoglobules. In this chapter, we will describe how the proteomic approaches have launched new perspectives on plastoglobule functions.

  18. NeuCode Proteomics Reveals Bap1 Regulation of Metabolism

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    Joshua M. Baughman

    2016-07-01

    Full Text Available We introduce neutron-encoded (NeuCode amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.

  19. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

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    Qingzhu Hua

    2016-09-01

    Full Text Available Red dragon fruit or red pitaya (Hylocereus polyrhizus is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to “phenylpropanoid biosynthesis”, “tyrosine metabolism”, “flavonoid biosynthesis”, “ascorbate and aldarate metabolism”, “betalains biosynthesis” and “anthocyanin biosynthesis”. In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

  20. Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal.

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    Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

    2015-05-18

    A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transformation. Cbx3 is up-regulated during gonad reversal and is likely to have a role in spermatogenesis. Rab37 is down-regulated during the reversal and is mainly associated with oogenesis. Both Cbx3 and Rab37 are linked up in a protein network. These datasets in gonadal proteomes provide a new resource for further studies in gonadal development.

  1. A proteomics study reveals a predominant change in MaoB expression in platelets of healthy volunteers after high protein meat diet

    DEFF Research Database (Denmark)

    Zellner, Maria; Babeluk, Rita; Jakobsen, Lene Holm

    2011-01-01

    Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP...... reproducibly studied platelet proteins only the level of monoamine oxidase B (MaoB), a neurotransmitter degrading enzyme, decreased by 26% significantly (adjusted P value diet. In addition, we found a correlation (r = 0.477; P ...) diet on both cognitive performance and corresponding biochemical parameters. A randomised intervention study was conducted with 23 healthy males (aged 19-31 years) to investigate the effects of a usual (UP) versus a HP diet on cognitive function and on the platelet proteome a well-established model...

  2. Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

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    Yuan, Peijia; D'Lima, Nadia G; Slavoff, Sarah A

    2018-01-09

    Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

  3. Principles of proteome allocation are revealed using proteomic data and genome-scale models

    DEFF Research Database (Denmark)

    Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.

    2016-01-01

    to metabolism and fitness. Using proteomics data, we formulated allocation constraints for key proteome sectors in the ME model. The resulting calibrated model effectively computed the "generalist" (wild-type) E. coli proteome and phenotype across diverse growth environments. Across 15 growth conditions......Integrating omics data to refine or make context-specific models is an active field of constraint-based modeling. Proteomics now cover over 95% of the Escherichia coli proteome by mass. Genome-scale models of Metabolism and macromolecular Expression (ME) compute proteome allocation linked...... of these sectors for the general stress response sigma factor sigma(S). Finally, the sector constraints represent a general formalism for integrating omics data from any experimental condition into constraint-based ME models. The constraints can be fine-grained (individual proteins) or coarse-grained (functionally...

  4. Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats

    International Nuclear Information System (INIS)

    Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

    2017-01-01

    Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. - Highlights: • Arsenic exposure has been associated with a number of adverse health effects. • The molecular mechanisms involved in arsenic-induced cardiotoxicity remain unclear. • Differential proteins were identified in arsenic-exposed rat heart by proteomics. • Arsenic induces heart toxicity through the Akt/p38 MAPK signaling pathway. - Label-free quantitative proteomic analysis of rat heart reveals putative mechanisms and biomarkers for arsenic-induced cardiotoxicity.

  5. Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

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    Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

    2017-01-01

    Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

  6. Single muscle fiber proteomics reveals unexpected mitochondrial specialization

    DEFF Research Database (Denmark)

    Murgia, Marta; Nagaraj, Nagarjuna; Deshmukh, Atul S

    2015-01-01

    and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions...

  7. Transcriptome and proteome data reveal candidate genes for pollinator attraction in sexually deceptive orchids.

    Science.gov (United States)

    Sedeek, Khalid E M; Qi, Weihong; Schauer, Monica A; Gupta, Alok K; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P; Schlüter, Philipp M

    2013-01-01

    Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of

  8. Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics

    DEFF Research Database (Denmark)

    Yang, Fen; Braga, Marcella Nunes de Melo; Larsen, Martin Røssel

    2013-01-01

    The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat-S. tritici interaction, we performed a time-course study of S. tritici infection...... in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during...... compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger...

  9. Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus

    Directory of Open Access Journals (Sweden)

    Ann Ray

    2016-07-01

    Full Text Available Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells.

  10. Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

    Directory of Open Access Journals (Sweden)

    Patricia eNAGNAN-LE MEILLOUR

    2014-12-01

    Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

  11. Data-Independent Acquisition-Based Quantitative Proteomic Analysis Reveals Potential Biomarkers of Kidney Cancer.

    Science.gov (United States)

    Song, Yimeng; Zhong, Lijun; Zhou, Juntuo; Lu, Min; Xing, Tianying; Ma, Lulin; Shen, Jing

    2017-12-01

    Renal cell carcinoma (RCC) is a malignant and metastatic cancer with 95% mortality, and clear cell RCC (ccRCC) is the most observed among the five major subtypes of RCC. Specific biomarkers that can distinguish cancer tissues from adjacent normal tissues should be developed to diagnose this disease in early stages and conduct a reliable prognostic evaluation. Data-independent acquisition (DIA) strategy has been widely employed in proteomic analysis because of various advantages, including enhanced protein coverage and reliable data acquisition. In this study, a DIA workflow is constructed on a quadrupole-Orbitrap LC-MS platform to reveal dysregulated proteins between ccRCC and adjacent normal tissues. More than 4000 proteins are identified, 436 of these proteins are dysregulated in ccRCC tissues. Bioinformatic analysis reveals that multiple pathways and Gene Ontology items are strongly associated with ccRCC. The expression levels of L-lactate dehydrogenase A chain, annexin A4, nicotinamide N-methyltransferase, and perilipin-2 examined through RT-qPCR, Western blot, and immunohistochemistry confirm the validity of the proteomic analysis results. The proposed DIA workflow yields optimum time efficiency and data reliability and provides a good choice for proteomic analysis in biological and clinical studies, and these dysregulated proteins might be potential biomarkers for ccRCC diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

    Science.gov (United States)

    Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

    2013-01-01

    Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

  13. Alanine Enhances Aminoglycosides-Induced ROS Production as Revealed by Proteomic Analysis

    Directory of Open Access Journals (Sweden)

    Jin-zhou Ye

    2018-01-01

    Full Text Available Metabolite-enabled killing of antibiotic-resistant pathogens by antibiotics is an attractive strategy to manage antibiotic resistance. Our previous study demonstrated that alanine or/and glucose increased the killing efficacy of kanamycin on antibiotic-resistant bacteria, whose action is through up-regulating TCA cycle, increasing proton motive force and enhancing antibiotic uptake. Despite the fact that alanine altered several metabolic pathways, other mechanisms could be potentially involved in alanine-mediated kanamycin killing of bacteria which remains to be explored. In the present study, we adopted proteomic approach to analyze the proteome changes induced by exogenous alanine. Our results revealed that the expression of three outer membrane proteins was altered and the deletion of nagE and fadL decreased the intracellular kanamycin concentration, implying their possible roles in mediating kanamycin transport. More importantly, the integrated analysis of proteomic and metabolomic data pointed out that alanine metabolism could connect to riboflavin metabolism that provides the source for reactive oxygen species (ROS production. Functional studies confirmed that alanine treatment together with kanamycin could promote ROS production that in turn potentiates the killing of antibiotic-resistant bacteria. Further investigation showed that alanine repressed the transcription of antioxidant-encoding genes, and alanine metabolism to riboflavin metabolism connected with riboflavin metabolism through TCA cycle, glucogenesis pathway and pentose phosphate pathway. Our results suggest a novel mechanism by which alanine facilitates kanamycin killing of antibiotic-resistant bacteria via promoting ROS production.

  14. Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

    Science.gov (United States)

    Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

    2017-09-01

    The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for

  15. Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

    Directory of Open Access Journals (Sweden)

    Jaiswal Dinesh Kumar

    2012-10-01

    Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

  16. Proteomics reveals the effects of sustained weight loss on the human plasma proteome

    DEFF Research Database (Denmark)

    Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

    2016-01-01

    Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed ...

  17. Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

    DEFF Research Database (Denmark)

    Gallo, G.; Renzone, G.; Alduina, R.

    2010-01-01

    A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially...... available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis...... intermediates, were upregulated during antibiotic production. qRT-PCR analysis revealed that 8 out of 14 upregulated genes showed a positive correlation between changes at translational and transcriptional expression level. Furthermore, proteomic analysis of two nonproducing mutants, restricted to a sub...

  18. Proteomic Technologies for the Study of Osteosarcoma

    Directory of Open Access Journals (Sweden)

    Stephanie D. Byrum

    2012-01-01

    Full Text Available Osteosarcoma is the most common primary bone cancer of children and is established during stages of rapid bone growth. The disease is a consequence of immature osteoblast differentiation, which gives way to a rapidly synthesized incompletely mineralized and disorganized bone matrix. The mechanism of osteosarcoma tumorogenesis is poorly understood, and few proteomic studies have been used to interrogate the disease thus far. Accordingly, these studies have identified proteins that have been known to be associated with other malignancies, rather than being osteosarcoma specific. In this paper, we focus on the growing list of available state-of-the-art proteomic technologies and their specific application to the discovery of novel osteosarcoma diagnostic and therapeutic targets. The current signaling markers/pathways associated with primary and metastatic osteosarcoma that have been identified by early-stage proteomic technologies thus far are also described.

  19. Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal

    OpenAIRE

    Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

    2015-01-01

    A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transf...

  20. Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

    Science.gov (United States)

    Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

    2014-12-01

    Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

    KAUST Repository

    Mu, Huawei; Sun, Jin; Cheung, Siu Gin; Fang, Ling; Zhou, Haiyun; Luan, Tiangang; Zhang, Huoming; Wong, Chris K.C.; Qiu, Jian-Wen

    2017-01-01

    Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic

  2. Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

    KAUST Repository

    Mu, Huawei

    2017-11-06

    Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic

  3. Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics.

    Science.gov (United States)

    Yang, Fen; Melo-Braga, Marcella N; Larsen, Martin R; Jørgensen, Hans J L; Palmisano, Giuseppe

    2013-09-01

    The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat-S. tritici interaction, we performed a time-course study of S. tritici infection in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger accumulation of signal molecules, including calcium, H2O2, NO, and sugars, in the resistant than in the susceptible cultivar in response to the infection. Additionally, 31 proteins and 5 phosphoproteins from the pathogen were identified, including metabolic proteins and signaling proteins such as GTP-binding proteins, 14-3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a comprehensive

  4. Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

    Directory of Open Access Journals (Sweden)

    Monique Ramos de Oliveira Trugilho

    2017-05-01

    Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

  5. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta "Gold Standard" Leaves.

    Science.gov (United States)

    Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

    2016-03-08

    Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta "Gold Standard" is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta "Gold Standard", as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta "Gold Standard". For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.

  6. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon.

    Science.gov (United States)

    Sousa, Leijiane F; Portes-Junior, José A; Nicolau, Carolina A; Bernardoni, Juliana L; Nishiyama, Milton Y; Amazonas, Diana R; Freitas-de-Sousa, Luciana A; Mourão, Rosa Hv; Chalkidis, Hipócrates M; Valente, Richard H; Moura-da-Silva, Ana M

    2017-04-21

    Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA 2 s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA 2 s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not

  7. Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

    International Nuclear Information System (INIS)

    Jami, Mohammad-Saeid; Huang, Xin; Peng, Hong; Fu, Kai; Li, Yan; Singh, Rakesh K; Ding, Shi-Jian; Hou, Jinxuan; Liu, Miao; Varney, Michelle L; Hassan, Hesham; Dong, Jixin; Geng, Liying; Wang, Jing; Yu, Fang

    2014-01-01

    KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it

  8. Proteomics in studying cancer stem cell biology.

    Science.gov (United States)

    Kranenburg, Onno; Emmink, Benjamin L; Knol, Jaco; van Houdt, Winan J; Rinkes, Inne H M Borel; Jimenez, Connie R

    2012-06-01

    Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.

  9. Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

    Science.gov (United States)

    Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

    2015-01-01

    Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

  10. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    DEFF Research Database (Denmark)

    Young, Jette Feveile; Larsen, Lotte Bach; Malmendal, Anders

    2010-01-01

    Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat......-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating...... the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. METHODS: Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension...

  11. Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

    2017-10-06

    Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

  12. Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication*

    Science.gov (United States)

    Kito, Keiji; Ito, Haruka; Nohara, Takehiro; Ohnishi, Mihoko; Ishibashi, Yuko; Takeda, Daisuke

    2016-01-01

    Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under

  13. Contrasting patterns of evolutionary constraint and novelty revealed by comparative sperm proteomic analysis in Lepidoptera.

    Science.gov (United States)

    Whittington, Emma; Forsythe, Desiree; Borziak, Kirill; Karr, Timothy L; Walters, James R; Dorus, Steve

    2017-12-02

    Rapid evolution is a hallmark of reproductive genetic systems and arises through the combined processes of sequence divergence, gene gain and loss, and changes in gene and protein expression. While studies aiming to disentangle the molecular ramifications of these processes are progressing, we still know little about the genetic basis of evolutionary transitions in reproductive systems. Here we conduct the first comparative analysis of sperm proteomes in Lepidoptera, a group that exhibits dichotomous spermatogenesis, in which males produce a functional fertilization-competent sperm (eupyrene) and an incompetent sperm morph lacking nuclear DNA (apyrene). Through the integrated application of evolutionary proteomics and genomics, we characterize the genomic patterns potentially associated with the origination and evolution of this unique spermatogenic process and assess the importance of genetic novelty in Lepidopteran sperm biology. Comparison of the newly characterized Monarch butterfly (Danaus plexippus) sperm proteome to those of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated conservation at the level of protein abundance and post-translational modification within Lepidoptera. In contrast, comparative genomic analyses across insects reveals significant divergence at two levels that differentiate the genetic architecture of sperm in Lepidoptera from other insects. First, a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insect species was observed. Second, a substantial number of sperm proteins were found to be specific to Lepidoptera, in that they lack detectable homology to the genomes of more distantly related insects. Lastly, the functional importance of Lepidoptera specific sperm proteins is broadly supported by their increased abundance relative to proteins conserved across insects. Our results identify a burst of genetic novelty

  14. Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

    Science.gov (United States)

    Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

    2016-06-30

    Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

  15. Systems biology integration of proteomic data in rodent models of depression reveals involvement of the immune response and glutamatergic signaling.

    Science.gov (United States)

    Carboni, Lucia; Nguyen, Thanh-Phuong; Caberlotto, Laura

    2016-12-01

    The pathophysiological basis of major depression is incompletely understood. Recently, numerous proteomic studies have been performed in rodent models of depression to investigate the molecular underpinnings of depressive-like behaviours with an unbiased approach. The objective of the study is to integrate the results of these proteomic studies in depression models to shed light on the most relevant molecular pathways involved in the disease. Network analysis is performed integrating preexisting proteomic data from rodent models of depression. The IntAct mouse and the HRPD are used as reference protein-protein interaction databases. The functionality analyses of the networks are then performed by testing overrepresented GO biological process terms and pathways. Functional enrichment analyses of the networks revealed an association with molecular processes related to depression in humans, such as those involved in the immune response. Pathways impacted by clinically effective antidepressants are modulated, including glutamatergic signaling and neurotrophic responses. Moreover, dysregulations of proteins regulating energy metabolism and circadian rhythms are implicated. The comparison with protein pathways modulated in depressive patients revealed significant overlapping. This systems biology study supports the notion that animal models can contribute to the research into the biology and therapeutics of depression. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Water deficit mechanisms in perennial shrubs Cerasus humilis leaves revealed by physiological and proteomic analyses.

    Science.gov (United States)

    Yin, Zepeng; Ren, Jing; Zhou, Lijuan; Sun, Lina; Wang, Jiewan; Liu, Yulong; Song, Xingshun

    2016-01-01

    Drought (Water deficit, WD) poses a serious threat to extensively economic losses of trees throughout the world. Chinese dwarf cherry ( Cerasus humilis ) is a good perennial plant for studying the physiological and sophisticated molecular network under WD. The aim of this study is to identify the effect of WD on C. humilis through physiological and global proteomics analysis and improve understanding of the WD resistance of plants. Currently, physiological parameters were applied to investigate C. humilis response to WD. Moreover, we used two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in C. humilis leaves subjected to WD (24 d). Furthermore, we also examined the correlation between protein and transcript levels. Several physiological parameters, including relative water content and Pn were reduced by WD. In addition, the malondialdehyde (MDA), relative electrolyte leakage (REL), total soluble sugar, and proline were increased in WD-treated C. humilis . Comparative proteomic analysis revealed 46 protein spots (representing 43 unique proteins) differentially expressed in C. humilis leaves under WD. These proteins were mainly involved in photosynthesis, ROS scavenging, carbohydrate metabolism, transcription, protein synthesis, protein processing, and nitrogen and amino acid metabolisms, respectively. WD promoted the CO 2 assimilation by increase light reaction and Calvin cycle, leading to the reprogramming of carbon metabolism. Moreover, the accumulation of osmolytes (i.e., proline and total soluble sugar) and enhancement of ascorbate-glutathione cycle and glutathione peroxidase/glutathione s-transferase pathway in leaves could minimize oxidative damage of membrane and other molecules under WD. Importantly, the regulation role of carbohydrate metabolisms (e. g. glycolysis, pentose phosphate pathways, and TCA) was enhanced. These findings provide key candidate proteins for genetic improvement of perennial plants metabolism under

  17. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    Science.gov (United States)

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  18. Physiological and Proteomics Analyses Reveal Low-Phosphorus Stress Affected the Regulation of Photosynthesis in Soybean.

    Science.gov (United States)

    Chu, Shanshan; Li, Hongyan; Zhang, Xiangqian; Yu, Kaiye; Chao, Maoni; Han, Suoyi; Zhang, Dan

    2018-06-06

    Previous studies have revealed a significant genetic relationship between phosphorus (P)-efficiency and photosynthesis-related traits in soybean. In this study, we used proteome profiling in combination with expression analysis, biochemical investigations, and leaf ultrastructural analysis to identify the underlying physiological and molecular responses. The expression analysis and ultrastructural analysis showed that the photosynthesis key genes were decreased at transcript levels and the leaf mesophyll and chloroplast were severely damaged after low-P stress. Approximately 55 protein spots showed changes under low-P condition by mass spectrometry, of which 17 were involved in various photosynthetic processes. Further analysis revealed the depression of photosynthesis caused by low-P stress mainly involves the regulation of leaf structure, adenosine triphosphate (ATP) synthesis, absorption and transportation of CO₂, photosynthetic electron transport, production of assimilatory power, and levels of enzymes related to the Calvin cycle. In summary, our findings indicated that the existence of a stringent relationship between P supply and the genomic control of photosynthesis in soybean. As an important strategy to protect soybean photosynthesis, P could maintain the stability of cell structure, up-regulate the enzymes’ activities, recover the process of photosystem II (PSII), and induce the expression of low-P responsive genes and proteins.

  19. Proteomic Characterization of Armillaria mellea Reveals Oxidative Stress Response Mechanisms and Altered Secondary Metabolism Profiles

    Directory of Open Access Journals (Sweden)

    Cassandra Collins

    2017-09-01

    Full Text Available Armillaria mellea is a major plant pathogen. Yet, the strategies the organism uses to infect susceptible species, degrade lignocellulose and other plant material and protect itself against plant defences and its own glycodegradative arsenal are largely unknown. Here, we use a combination of gel and MS-based proteomics to profile A. mellea under conditions of oxidative stress and changes in growth matrix. 2-DE and LC-MS/MS were used to investigate the response of A. mellea to H2O2 and menadione/FeCl3 exposure, respectively. Several proteins were detected with altered abundance in response to H2O2, but not menadione/FeCl3 (i.e., valosin-containing protein, indicating distinct responses to these different forms of oxidative stress. One protein, cobalamin-independent methionine synthase, demonstrated a common response in both conditions, which may be a marker for a more general stress response mechanism. Further changes to the A. mellea proteome were investigated using MS-based proteomics, which identified changes to putative secondary metabolism (SM enzymes upon growth in agar compared to liquid cultures. Metabolomic analyses revealed distinct profiles, highlighting the effect of growth matrix on SM production. This establishes robust methods by which to utilize comparative proteomics to characterize this important phytopathogen.

  20. The distinctive gastric fluid proteome in gastric cancer reveals a multi-biomarker diagnostic profile

    Directory of Open Access Journals (Sweden)

    Eng Alvin KH

    2008-10-01

    Full Text Available Abstract Background Overall gastric cancer survival remains poor mainly because there are no reliable methods for identifying highly curable early stage disease. Multi-protein profiling of gastric fluids, obtained from the anatomic site of pathology, could reveal diagnostic proteomic fingerprints. Methods Protein profiles were generated from gastric fluid samples of 19 gastric cancer and 36 benign gastritides patients undergoing elective, clinically-indicated gastroscopy using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on multiple ProteinChip arrays. Proteomic features were compared by significance analysis of microarray algorithm and two-way hierarchical clustering. A second blinded sample set (24 gastric cancers and 29 clinically benign gastritides was used for validation. Results By significance analysyis of microarray, 60 proteomic features were up-regulated and 46 were down-regulated in gastric cancer samples (p Conclusion This simple and reproducible multimarker proteomic assay could supplement clinical gastroscopic evaluation of symptomatic patients to enhance diagnostic accuracy for gastric cancer and pre-malignant lesions.

  1. Targeted proteomics guided by label-free global proteome analysis in saliva reveal transition signatures from health to periodontal disease.

    Science.gov (United States)

    Bostanci, Nagihan; Selevsek, Nathalie; Wolski, Witold; Grossmann, Jonas; Bao, Kai; Wahlander, Asa; Trachsel, Christian; Schlapbach, Ralph; Özturk, Veli Özgen; Afacan, Beral; Emingil, Gulnur; Belibasakis, Georgios N

    2018-04-02

    Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. We carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n=67, health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n=82). The LFQ platform led to the discovery of 119 proteins with at least two-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1), with maximum area under the receiver operating curve >0.97. This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum

  2. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  3. A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

    Science.gov (United States)

    Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

    2018-03-19

    Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to

  4. Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas.

    Science.gov (United States)

    Timmins-Schiffman, Emma; Coffey, William D; Hua, Wilber; Nunn, Brook L; Dickinson, Gary H; Roberts, Steven B

    2014-11-03

    Ocean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end-of-century open ocean pH reductions. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different p CO2 levels for four weeks: 400 μatm (pH 8.0), 800 μatm (pH 7.7), 1000 μatm (pH 7.6), or 2800 μatm (pH 7.3). At the end of the four week exposure period, oysters in all four p CO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated p CO2. Elevated p CO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with p CO2, with numerous processes significantly affected by mechanical stimulation at high versus low p CO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835). Oyster physiology is significantly altered by exposure to elevated p CO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of p CO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.

  5. Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

    Directory of Open Access Journals (Sweden)

    Nicos Angelopoulos

    2016-06-01

    Full Text Available Tyrosine kinases (TKs play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015 [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015 [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

  6. Differential proteomics reveals the hallmarks of seed development in common bean (Phaseolus vulgaris L.)

    NARCIS (Netherlands)

    Parreira, J R; Bouraada, J; Fitzpatrick, M A; Silvestre, S; Bernardes da Silva, A; Marques da Silva, J; Almeida, A M; Fevereiro, P; Altelaar, A F M; Araújo, S S

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is one of the most consumed staple foods worldwide. Little is known about the molecular mechanisms controlling seed development. This study aims to comprehensively describe proteome dynamics during seed development of common bean. A high-throughput gel-free

  7. Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer*

    Science.gov (United States)

    Chen, Chien-Lun; Chung, Ting; Wu, Chih-Ching; Ng, Kwai-Fong; Yu, Jau-Song; Tsai, Cheng-Han; Chang, Yu-Sun; Liang, Ying; Tsui, Ke-Hung; Chen, Yi-Ting

    2015-01-01

    More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins—SLC3A2, STMN1, and TAGLN2—in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant

  8. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  9. Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

    Directory of Open Access Journals (Sweden)

    Candida Vannini

    Full Text Available Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

  10. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  11. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

    Science.gov (United States)

    Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

    2014-10-01

    Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

  12. Proteomic analysis reveals changes in carbohydrate and protein metabolism associated with broiler breast myopathy.

    Science.gov (United States)

    Kuttappan, Vivek A; Bottje, Walter; Ramnathan, Ranjith; Hartson, Steven D; Coon, Craig N; Kong, Byung-Whi; Owens, Casey M; Vazquez-Añon, Mercedes; Hargis, Billy M

    2017-08-01

    White Striping (WS) and Woody Breast (WB) are 2 conditions that adversely affect consumer acceptance as well as quality of poultry meat and meat products. Both WS and WB are characterized with degenerative myopathic changes. Previous studies showed that WS and WB in broiler fillets could result in higher ultimate pH, increased drip loss, and decreased marinade uptake. The main objective of the present study was to compare the proteomic profiles of muscle tissue (n = 5 per group) with either NORM (no or few minor myopathic lesions) or SEV (with severe myopathic changes). Proteins were extracted from these samples and analyzed using a hybrid LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). Over 800 proteins were identified in the muscle samples, among which 141 demonstrated differential (P < 0.05) expression between NORM and SEV. The set of differentially (P < 0.05) expressed proteins was uploaded to Ingenuity Pathway Analysis® (IPA) software to determine the associated biological networks and pathways. The IPA analysis showed that eukaryotic initiation factor-2 (eIF-2) signaling, mechanistic target of rapamycin (mTOR) signaling, as well as regulation of eIF4 and p70S6K signaling were the major canonical pathways up-regulated (P < 0.05) in SEV muscle compared to NORM. The up-regulation of these pathways indicate an increase in protein synthesis which could be part of the rapid growth as well as cellular stress associated with ongoing muscle degeneration and the attempt to repair tissue damage in SEV birds. Furthermore, IPA analysis revealed that glycolysis and gluconeogenesis were the major down-regulated (P < 0.05) canonical pathways in SEV with respect to NORM muscle. Down-regulation of these pathways could be the reason for higher ultimate pH seen in SEV muscle samples indicating reduced glycolytic potential. In conclusion, comparison of proteomic profiles of NORM and SEV muscle samples showed differences in protein profile which explains some of the observed

  13. Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Aranjuelo, Iker; Molero, Gemma; Erice, Gorka; Avice, Jean Christophe; Nogués, Salvador

    2011-01-01

    Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation

  14. Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol.

    Science.gov (United States)

    Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2013-01-14

    Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse phosphorus stress response.

    Directory of Open Access Journals (Sweden)

    Sonya T Dyhrman

    Full Text Available Phosphorus (P is a critical driver of phytoplankton growth and ecosystem function in the ocean. Diatoms are an abundant class of marine phytoplankton that are responsible for significant amounts of primary production. With the control they exert on the oceanic carbon cycle, there have been a number of studies focused on how diatoms respond to limiting macro and micronutrients such as iron and nitrogen. However, diatom physiological responses to P deficiency are poorly understood. Here, we couple deep sequencing of transcript tags and quantitative proteomics to analyze the diatom Thalassiosira pseudonana grown under P-replete and P-deficient conditions. A total of 318 transcripts were differentially regulated with a false discovery rate of <0.05, and a total of 136 proteins were differentially abundant (p<0.05. Significant changes in the abundance of transcripts and proteins were observed and coordinated for multiple biochemical pathways, including glycolysis and translation. Patterns in transcript and protein abundance were also linked to physiological changes in cellular P distributions, and enzyme activities. These data demonstrate that diatom P deficiency results in changes in cellular P allocation through polyphosphate production, increased P transport, a switch to utilization of dissolved organic P through increased production of metalloenzymes, and a remodeling of the cell surface through production of sulfolipids. Together, these findings reveal that T. pseudonana has evolved a sophisticated response to P deficiency involving multiple biochemical strategies that are likely critical to its ability to respond to variations in environmental P availability.

  16. Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

    Science.gov (United States)

    Zai, Xiaodong; Yang, Qiaoling; Yin, Ying; Li, Ruihua; Qian, Mengying; Zhao, Taoran; Li, Yaohui; Zhang, Jun; Fu, Ling; Xu, Junjie; Chen, Wei

    2017-01-01

    Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic

  17. Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

    Directory of Open Access Journals (Sweden)

    Xiaodong Zai

    2017-11-01

    Full Text Available Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular

  18. iTRAQ-based proteomic analysis reveals alterations in the liver induced by restricted meal frequency in a pig model.

    Science.gov (United States)

    Liu, Jingbo; Liu, Zhengqun; Chen, Liang; Zhang, Hongfu

    2016-01-01

    The present study was conducted to investigate the effects of meal frequency on metabolite levels in pig plasma and hepatic proteome by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Twenty-four pigs (60.7 ± 1.0 kg) consumed the same amount of feed either in 2 (M2, n = 12) or 12 (M12, n = 12) meals per day. After an 8-wk feeding period, plasma concentrations of metabolites and hormones, hepatic biochemical traits, and proteome (n = 4 per group) were measured. Pigs on the M12 regimen had lower average daily gain and gain-to-feed ratio than pigs fed the M2 regimen. The M2 regimen resulted in lower total lipid, glycogen, and triacylglycerol content in the liver and circulating triacylglycerol concentration than that in the M12 pigs. The metabolic hormone concentrations were not affected by meal frequency, with the exception of elevated fibroblast growth factor 21 concentrations in the M2 regimen compared with the M12 regimen. The iTRAQ-based proteomic analysis revealed 35 differentially expressed proteins in the liver between pigs fed two and 12 meals per day, and these differentially expressed proteins were involved in the regulation of general biological process such as glucose and energy metabolism, lipid metabolism, protein and amino acid metabolism, stress response, and cell redox homeostasis. Altogether, the proteomic results provide insights into the mechanism mediating the beneficial effects of restricted meal frequency on the metabolic fitness. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

    Science.gov (United States)

    Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

    2011-10-01

    The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

  20. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    Directory of Open Access Journals (Sweden)

    Nielsen Niels

    2010-02-01

    Full Text Available Abstract Background Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. Methods Differentiated mouse myotube cultures (C2C12 were exposed to 5 mM creatine monohydrate (CMH for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. Results The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the

  1. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

    OpenAIRE

    Wang, Yong-Qiang; Yang, Yong; Fei, Zhangjun; Yuan, Hui; Fish, Tara; Thannhauser, Theodore W.; Mazourek, Michael; Kochian, Leon V.; Wang, Xiaowu; Li, Li

    2013-01-01

    Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953?2262 proteins from chromoplasts of diff...

  2. Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Maria Liguori

    Full Text Available The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF proteomic profiles of Multiple Sclerosis (MS patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS, 16 Relapsing Remitting (RR MS, 11 Progressive (Pr MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000-25000 Da. Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥ 1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05, whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04. Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013. Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS.

  3. Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

    Science.gov (United States)

    Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

    2010-12-01

    Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

  4. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

  5. Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats

    DEFF Research Database (Denmark)

    Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen

    2017-01-01

    Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative...... proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33...... proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb...

  6. Alteration of cystatin C in cerebrospinal fluid of patients with sciatica revealed by a proteomical approach

    International Nuclear Information System (INIS)

    Liu, X; Zeng, B.; Xu, J.

    2005-01-01

    To better understand the pathophysiological mechanisms underlying sciatica induced by lumbar intervertebral disk herniation and to ascertain the protein that presents with the most observable changes in the cerebrospinal fluid (CSF) of patients with sciatica. We conducted the study in the Key Laboratory of Shanghai 6th People's Hospital, Shanghai Jiaotong University, Shanghai, Peoples Republic of China, during the period June 2004 to March 2005. In 2 separate experiments, we carried out the study involving the CSF of sciatica patients (the case group) and the CSF of otherwise, healthy volunteers (the control group). We utilized a proteomical analysis to compare the samples of 10 patients with sciatica with 10 volunteers in the control group. We individually separated each of the groups' CSF by 2-dimensional gel electrophoresis. We analyzed the harvested gel images with PD Quest 2D-gel software (Bio-Rad) to ascertain the differential proteins between the 2 groups. We based the enzyme linked immuno- absorbent assay (ELISA) experiment, which followed, on the results of the first experiment. We found 15 of the protein spots in the CSF differed appreciably in varying degrees between the 2 groups, and identification made by LC-MS/MS revealed that the most significant disparity was with cystatin C. The result of ELISA experiment confirmed a considerable decrease in the level of cystatin C (p<0.01) in the patients with sciatica. In the CSF of patients with sciatica, the volume of cystatin C increased markedly indicating that it may play an important role in the pathophysiological processes of sciatica. (author)

  7. Proteomic Profiles Reveal the Function of Different Vegetative Tissues of Moringa oleifera.

    Science.gov (United States)

    Wang, Lei; Zou, Qiong; Wang, Jinxing; Zhang, Junjie; Liu, Zeping; Chen, Xiaoyang

    2016-12-01

    Moringa oleifera is a rich source of bioactive compounds and is widely used in traditional medicine and food for its nutritional value; however, the protein and peptide components of different tissues are rarely discussed. Here, we describe the first investigation of M. oleifera proteomes using mass spectrometry and bioinformatics methods. We aimed to elucidate the protein profiles of M. oleifera leaves, stem, bark, and root. Totally 202 proteins were identified from four vegetative organs. We identified 101 proteins from leaves, 51 from stem, 94 from bark and 67 from root, finding that only five proteins existed in both four vegetative parts. The calculated pI of most of the proteins is distributed in 5-10 and the molecular weight distributed below 100 kDa. Functional classification analysis revealed that proteins which are involved in catalytic activities are the most abundant both in leaves, stem, bark and root. Identification of several heat shock proteins in four vegetative tissues might be adaptive for resistance to high temperature environmental stresses of tropical or subtropical areas. Some enzymes involved in antioxidant processes were also identified in M. oleifera leaves, stem, bark and root. Among the four tissues studies here, leaves protein content and molecular diversity were the highest. The identification of the flocculating protein MO2.1 and MO2.2 in the bark and root provides clue to clarify the antimicrobial molecular mechanisms of root and bark. This study provides information on the protein compositions of M. oleifera vegetative tissues that will be beneficial for potential drug and food supplement development and plant physiology research.

  8. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development.

    Science.gov (United States)

    Wang, Yong-Qiang; Yang, Yong; Fei, Zhangjun; Yuan, Hui; Fish, Tara; Thannhauser, Theodore W; Mazourek, Michael; Kochian, Leon V; Wang, Xiaowu; Li, Li

    2013-02-01

    Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953-2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.

  9. Gel-free proteomics reveal potential biomarkers of priming-induced salt tolerance in durum wheat.

    Science.gov (United States)

    Fercha, Azzedine; Capriotti, Anna Laura; Caruso, Giuseppe; Cavaliere, Chiara; Gherroucha, Hocine; Samperi, Roberto; Stampachiacchiere, Serena; Lagana, Aldo

    2013-10-08

    Seed priming has been successfully demonstrated to be an efficient method to improve crop productivity under stressful conditions. As a first step toward better understanding of the mechanisms underlying the priming-induced salt stress tolerance in durum wheat, and to overcome the limitations of the gel-based approach, a comparative gel-free proteomic analysis was conducted with durum wheat seed samples of varying vigor as generated by hydro- and ascorbate-priming treatments. Results indicate that hydro-priming was accompanied by significant changes of 72 proteins, most of which are involved in proteolysis, protein synthesis, metabolism and disease/defense response. Ascorbate-priming was, however, accompanied by significant changes of 83 proteins, which are mainly involved in protein metabolism, antioxidant protection, repair processes and, interestingly, in methionine-related metabolism. The present study provides new information for understanding how 'priming-memory' invokes seed stress tolerance. The current work describes the first study in which gel-free shotgun proteomics were used to investigate the metabolic seed protein fraction in durum wheat. A combined approach of protein fractionation, hydrogel nanoparticle enrichment technique, and gel-free shotgun proteomic analysis allowed us to identify over 380 proteins exhibiting greater molecular weight diversity (ranging from 7 to 258kDa). Accordingly, we propose that this approach could be useful to acquire a wider perspective and a better understanding of the seed proteome. In the present work, we employed this method to investigate the potential biomarkers of priming-induced salt tolerance in durum wheat. In this way, we identified several previously unrecognized proteins which were never been reported before, particularly for the ascorbate-priming treatment. These findings could provide new avenues for improving crop productivity, particularly under unfavorable environmental conditions. © 2013.

  10. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

    2014-01-01

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

    KAUST Repository

    Zhu, Jiang

    2014-06-11

    Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. In vivo Host-Pathogen Interaction as Revealed by Global Proteomic Profiling of Zebrafish Larvae

    Directory of Open Access Journals (Sweden)

    Francisco Díaz-Pascual

    2017-07-01

    Full Text Available The outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1 and the host (zebrafish. We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, were exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, and cellular responses to antibiotic and starvation were enriched exclusively after exposure by

  13. Time-Series Analyses of Transcriptomes and Proteomes Reveal Molecular Networks Underlying Oil Accumulation in Canola.

    Science.gov (United States)

    Wan, Huafang; Cui, Yixin; Ding, Yijuan; Mei, Jiaqin; Dong, Hongli; Zhang, Wenxin; Wu, Shiqi; Liang, Ying; Zhang, Chunyu; Li, Jiana; Xiong, Qing; Qian, Wei

    2016-01-01

    Understanding the regulation of lipid metabolism is vital for genetic engineering of canola ( Brassica napus L.) to increase oil yield or modify oil composition. We conducted time-series analyses of transcriptomes and proteomes to uncover the molecular networks associated with oil accumulation and dynamic changes in these networks in canola. The expression levels of genes and proteins were measured at 2, 4, 6, and 8 weeks after pollination (WAP). Our results show that the biosynthesis of fatty acids is a dominant cellular process from 2 to 6 WAP, while the degradation mainly happens after 6 WAP. We found that genes in almost every node of fatty acid synthesis pathway were significantly up-regulated during oil accumulation. Moreover, significant expression changes of two genes, acetyl-CoA carboxylase and acyl-ACP desaturase, were detected on both transcriptomic and proteomic levels. We confirmed the temporal expression patterns revealed by the transcriptomic analyses using quantitative real-time PCR experiments. The gene set association analysis show that the biosynthesis of fatty acids and unsaturated fatty acids are the most significant biological processes from 2-4 WAP and 4-6 WAP, respectively, which is consistent with the results of time-series analyses. These results not only provide insight into the mechanisms underlying lipid metabolism, but also reveal novel candidate genes that are worth further investigation for their values in the genetic engineering of canola.

  14. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Directory of Open Access Journals (Sweden)

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  15. Analysis of biostimulated microbial communities from two field experiments reveals temporal and spatial differences in proteome profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, S.J.; Wilkins, M.J.; Nicora, C.D.; Williams, K.H.; Banfield, J.F.; VerBerkmoes, N.C.; Hettich, R.L.; NGuessan, A.L.; Mouser, P.J.; Elifantz, H.; Smith, R.D.; Lovley, D.R.; Lipton, M.S.; Long, P.E.

    2010-07-15

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetate-amended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or “pseudo-metagenomes”, for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally, a shift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  16. Proteomic and comparative genomic analysis reveals adaptability of Brassica napus to phosphorus-deficient stress.

    Science.gov (United States)

    Chen, Shuisen; Ding, Guangda; Wang, Zhenhua; Cai, Hongmei; Xu, Fangsen

    2015-03-18

    Given low solubility and immobility in many soils of the world, phosphorus (P) may be the most widely studied macronutrient for plants. In an attempt to gain an insight into the adaptability of Brassica napus to P deficiency, proteome alterations of roots and leaves in two B. napus contrasting genotypes, P-efficient 'Eyou Changjia' and P-inefficient 'B104-2', under long-term low P stress and short-term P-free starvation conditions were investigated, and proteomic combined with comparative genomic analyses were conducted to interpret the interrelation of differential abundance protein species (DAPs) responding to P deficiency with quantitative trait loci (QTLs) for P deficiency tolerance. P-efficient 'Eyou Changjia' had higher dry weight and P content, and showed high tolerance to low P stress compared with P-inefficient 'B104-2'. A total of 146 DAPs were successfully identified by MALDI TOF/TOF MS, which were categorized into several groups including defense and stress response, carbohydrate and energy metabolism, signaling and regulation, amino acid and fatty acid metabolism, protein process, biogenesis and cellular component, and function unknown. 94 of 146 DAPs were mapped to a linkage map constructed by a B. napus population derived from a cross between the two genotypes, and 72 DAPs were located in the confidence intervals of QTLs for P efficiency related traits. We conclude that the identification of these DAPs and the co-location of DAPs with QTLs in the B. napus linkage genetic map provide us novel information in understanding the adaptability of B. napus to P deficiency, and helpful to isolate P-efficient genes in B. napus. Low P seriously limits the production and quality of B. napus. Proteomics and genetic linkage map were widely used to study the adaptive strategies of B. napus response to P deficiency, proteomic combined with comparative genetic analysis to investigate the correlations between DAPs and QTLs are scarce. Thus, we herein investigated

  17. Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

    Energy Technology Data Exchange (ETDEWEB)

    Swelm, Rachel P.L. van [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Laarakkers, Coby M.M. [Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Pertijs, Jeanne C.L.M.; Verweij, Vivienne; Masereeuw, Rosalinde [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Russel, Frans G.M., E-mail: F.Russel@pharmtox.umcn.nl [Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen (Netherlands)

    2013-06-01

    Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75 mg/kg bw DF by oral gavage and 24 h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p < 0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p < 0.001 and p < 0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p < 0.001) and protein expression of PCNA (p < 0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24 h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury. - Highlights: • The urinary proteome shows biological processes involved in adverse drug reactions. • Urine proteins of DF-treated mice relate to kidney injury rather than liver injury. • Liver regeneration, not liver injury, is apparent 24h after oral DF administration. • Pretreatment with LPS does not enhance DF-induced liver injury in mice.

  18. Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

    Science.gov (United States)

    Tikka, Saara; Monogioudi, Evanthia; Gotsopoulos, Athanasios; Soliymani, Rabah; Pezzini, Francesco; Scifo, Enzo; Uusi-Rauva, Kristiina; Tyynelä, Jaana; Baumann, Marc; Jalanko, Anu; Simonati, Alessandro; Lalowski, Maciej

    2016-03-01

    Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood

  19. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells

    DEFF Research Database (Denmark)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

    2016-01-01

    to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...

  20. Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

    Science.gov (United States)

    Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

    2016-01-01

    Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

  1. Proteomic investigation of aphid honeydew reveals an unexpected diversity of proteins.

    Directory of Open Access Journals (Sweden)

    Ahmed Sabri

    Full Text Available Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora. Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu, and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective.

  2. Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

    Science.gov (United States)

    Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

    2014-12-05

    Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

  3. Proteomic analysis reveals contrasting stress response to uranium in two nitrogen-fixing Anabaena strains, differentially tolerant to uranium

    Energy Technology Data Exchange (ETDEWEB)

    Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar, E-mail: aptesk@barc.gov.in

    2017-01-15

    Highlights: • Response of two native cyanobacterial strains to uranium exposure was studied. • Anabaena L-31 exhibited higher tolerance to uranium as compared to Anabaena 7120. • Uranium exposure differentially affected the proteome profiles of the two strains. • Anabaena L-31 showed better sustenance of photosynthesis and carbon metabolism. • Anabaena L-31 displayed superior oxidative stress defense than Anabaena 7120. - Abstract: Two strains of the nitrogen-fixing cyanobacterium Anabaena, native to Indian paddy fields, displayed differential sensitivity to exposure to uranyl carbonate at neutral pH. Anabaena sp. strain PCC 7120 and Anabaena sp. strain L-31 displayed 50% reduction in survival (LD{sub 50} dose), following 3 h exposure to 75 μM and 200 μM uranyl carbonate, respectively. Uranium responsive proteome alterations were visualized by 2D gel electrophoresis, followed by protein identification by MALDI-ToF mass spectrometry. The two strains displayed significant differences in levels of proteins associated with photosynthesis, carbon metabolism, and oxidative stress alleviation, commensurate with their uranium tolerance. Higher uranium tolerance of Anabaena sp. strain L-31 could be attributed to sustained photosynthesis and carbon metabolism and superior oxidative stress defense, as compared to the uranium sensitive Anabaena sp. strain PCC 7120. Significance: Uranium responsive proteome modulations in two nitrogen-fixing strains of Anabaena, native to Indian paddy fields, revealed that rapid adaptation to better oxidative stress management, and maintenance of metabolic and energy homeostasis underlies superior uranium tolerance of Anabaena sp. strain L-31 compared to Anabaena sp. strain PCC 7120.

  4. The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats

    Directory of Open Access Journals (Sweden)

    Xu Yu-Dong

    2010-08-01

    Full Text Available Abstract Background The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR, which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma. Methods In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism. Results In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses. Conclusions Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

  5. Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

    Directory of Open Access Journals (Sweden)

    Hong-Jhang Chen

    Full Text Available Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD, a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877, 1.71% (15/877, and 2.62% (23/877 of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB (Alb, Fga, and Trf, suppressed excitotoxicity (Grm5, Gnai, and Gdi, and enhanced energy metabolism (Bdh, thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3 and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.

  6. Major depressive disorder: insight into candidate cerebrospinal fluid protein biomarkers from proteomics studies.

    Science.gov (United States)

    Al Shweiki, Mhd Rami; Oeckl, Patrick; Steinacker, Petra; Hengerer, Bastian; Schönfeldt-Lecuona, Carlos; Otto, Markus

    2017-06-01

    Major Depressive Disorder (MDD) is the leading cause of global disability, and an increasing body of literature suggests different cerebrospinal fluid (CSF) proteins as biomarkers of MDD. The aim of this review is to summarize the suggested CSF biomarkers and to analyze the MDD proteomics studies of CSF and brain tissues for promising biomarker candidates. Areas covered: The review includes the human studies found by a PubMed search using the following terms: 'depression cerebrospinal fluid biomarker', 'major depression biomarker CSF', 'depression CSF biomarker', 'proteomics depression', 'proteomics biomarkers in depression', 'proteomics CSF biomarker in depression', and 'major depressive disorder CSF'. The literature analysis highlights promising biomarker candidates and demonstrates conflicting results on others. It reveals 42 differentially regulated proteins in MDD that were identified in more than one proteomics study. It discusses the diagnostic potential of the biomarker candidates and their association with the suggested pathologies. Expert commentary: One ultimate goal of finding biomarkers for MDD is to improve the diagnostic accuracy to achieve better treatment outcomes; due to the heterogeneous nature of MDD, using bio-signatures could be a good strategy to differentiate MDD from other neuropsychiatric disorders. Notably, further validation studies of the suggested biomarkers are still needed.

  7. [Application progress of proteomic in pharmacological study of Chinese medicinal formulae].

    Science.gov (United States)

    Liu, Yu-Qian; Zhan, Shu-Yu; Ruan, Yu-Er; Zuo, Zhi-Yan; Ji, Xiao-Ming; Wang, Shuai-Jie; Ding, Bao-Yue

    2017-10-01

    Chinese medicinal formulae are the important means of clinical treatment in traditional Chinese medicine. It is urgent to use modern advanced scientific and technological means to reveal the complicated mechanism of Chinese medicinal formulae because they have the function characteristics of multiple components, multiple targets and integrated regulation. The systematic and comprehensive research model of proteomic is in line with the function characteristics of Chinese medicinal formulae, and proteomic has been widely used in the study of pharmacological mechanism of Chinese medicinal formulae. The recent applications of proteomic in pharmacological study of Chinese medicinal formulae in anti-cardiovascular and cerebrovascular diseases, anti-liver disease, antidiabetic, anticancer, anti-rheumatoid arthritis and other diseases were reviewed in this paper, and then the future development direction of proteomic in pharmacological study of Chinese medicinal formulae was put forward. This review is to provide the ideas and method for proteomic research on function mechanism of Chinese medicinal formulae. Copyright© by the Chinese Pharmaceutical Association.

  8. Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

    Science.gov (United States)

    Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

    2017-02-15

    Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

  9. Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics.

    Science.gov (United States)

    Kumar, Yashwant; Zhang, Limin; Panigrahi, Priyabrata; Dholakia, Bhushan B; Dewangan, Veena; Chavan, Sachin G; Kunjir, Shrikant M; Wu, Xiangyu; Li, Ning; Rajmohanan, Pattuparambil R; Kadoo, Narendra Y; Giri, Ashok P; Tang, Huiru; Gupta, Vidya S

    2016-07-01

    Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  10. 2D-DIGE analysis of mango (Mangifera indica L.) fruit reveals major proteomic changes associated with ripening.

    Science.gov (United States)

    Andrade, Jonathan de Magalhães; Toledo, Tatiana Torres; Nogueira, Silvia Beserra; Cordenunsi, Beatriz Rosana; Lajolo, Franco Maria; do Nascimento, João Roberto Oliveira

    2012-06-18

    A comparative proteomic investigation between the pre-climacteric and climacteric mango fruits (cv. Keitt) was performed to identify protein species with variable abundance during ripening. Proteins were phenol-extracted from fruits, cyanine-dye-labeled, and separated on 2D gels at pH 4-7. Total spot count of about 373 proteins spots was detected in each gel and forty-seven were consistently different between pre-climacteric and climacteric fruits and were subjected to LC-MS/MS analysis. Functional classification revealed that protein species involved in carbon fixation and hormone biosynthesis decreased during ripening, whereas those related to catabolism and the stress-response, including oxidative stress and abiotic and pathogen defense factors, accumulated. In relation to fruit quality, protein species putatively involved in color development and pulp softening were also identified. This study on mango proteomics provides an overview of the biological processes that occur during ripening. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Proteomics analysis of dendritic cell activation by contact allergens reveals possible biomarkers regulated by Nrf2

    Energy Technology Data Exchange (ETDEWEB)

    Mussotter, Franz, E-mail: franz.mussotter@bfr.bund.de [German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Berlin (Germany); Tomm, Janina Melanie [Helmholtz Centre for Environmental Research (UFZ), Department of Molecular Systems Biology, Leipzig (Germany); El Ali, Zeina; Pallardy, Marc; Kerdine-Römer, Saadia [INSERM UMR 996, Univ Paris-Sud, Université Paris-Saclay, Chátenay-Malabry (France); Götz, Mario [German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Berlin (Germany); Bergen, Martin von [Helmholtz Centre for Environmental Research (UFZ), Department of Molecular Systems Biology, Leipzig (Germany); University of Leipzig, Institute of Biochemistry, Leipzig (Germany); Aalborg University, Department of Chemistry and Bioscience, Aalborg (Denmark); Haase, Andrea; Luch, Andreas [German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Berlin (Germany)

    2016-12-15

    Allergic contact dermatitis is a widespread disease with high clinical relevance affecting approximately 20% of the general population. Typically, contact allergens are low molecular weight electrophilic compounds which can activate the Keap1/Nrf2 pathway. We performed a proteomics study to reveal possible biomarkers for dendritic cell (DC) activation by contact allergens and to further elucidate the role of Keap1/Nrf2 signaling in this process. We used bone marrow derived dendritic cells (BMDCs) of wild-type (nrf2{sup +/+}) and Nrf2 knockout (nrf2{sup −/−}) mice and studied their response against the model contact sensitizers 2,4-dinitrochlorobenzene (DNCB), cinnamaldehyde (CA) and nickel(II) sulfate by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS). Sodium dodecyl sulfate (SDS, 100 μM) served as irritant control. While treatment with nickel(II) sulfate and SDS had only little effects, CA and DNCB led to significant changes in protein expression. We found 18 and 30 protein spots up-regulated in wild-type cells treated with 50 and 100 μM CA, respectively. For 5 and 10 μM DNCB, 32 and 37 spots were up-regulated, respectively. Almost all of these proteins were not differentially expressed in nrf2{sup −/−} BMDCs, indicating an Nrf2-dependent regulation. Among them proteins were detected which are involved in oxidative stress and heat shock responses, as well as in signal transduction or basic cellular pathways. The applied approach allowed us to differentiate between Nrf2-dependent and Nrf2-independent cellular biomarkers differentially regulated upon allergen-induced DC activation. The data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. - Highlights: • Contact allergens induce proteins involved in DC maturation Nrf2-dependently. • Induction of these proteins points to a functional

  12. Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

    Science.gov (United States)

    Keller, Lani C; Romijn, Edwin P; Zamora, Ivan; Yates, John R; Marshall, Wallace F

    2005-06-21

    The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

  13. Lessons from the proteomic study of osteoarthritis.

    Science.gov (United States)

    Ruiz-Romero, Cristina; Fernández-Puente, Patricia; Calamia, Valentina; Blanco, Francisco J

    2015-08-01

    Osteoarthritis is the most common rheumatic pathology and one of the leading causes of disability worldwide. It is a very complex disease whose etiopathogenesis is not fully understood. Furthermore, there are serious limitations for its management, since it lacks specific and sensitive biomarkers for early diagnosis, prognosis and therapeutic monitoring. Proteomic approaches performed in the last few decades have contributed to the knowledge on the molecular mechanisms that participate in this pathology and they have also led to interesting panels of putative biomarker candidates. In the next few years, further efforts should be made for translating these findings into the clinical routines. It is expected that targeted proteomics strategies will be highly valuable for the verification and qualification of biomarkers of osteoarthritis.

  14. Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

    Science.gov (United States)

    Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

    2016-02-15

    The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    2013-01-01

    Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  16. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

    2013-01-22

    Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  17. Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

    Directory of Open Access Journals (Sweden)

    Aayudh Das

    2016-01-01

    Full Text Available Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

  18. Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

    Science.gov (United States)

    Gao, Yanpan; Chen, Yanyu; Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

    2017-01-31

    Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

  19. Nucleophosmin in the pathogenesis of arsenic-related bladder carcinogenesis revealed by quantitative proteomics

    International Nuclear Information System (INIS)

    Chen Shuhui; Wang Yiwen; Hsu Jueliang; Chang Hongyi; Wang Chiyun; Shen Potsun; Chiang Chiwu; Chuang Jingjing; Tsai Hungwen; Gu Powen; Chang Fangchih; Liu Hsiaosheng; Chow Nanhaw

    2010-01-01

    To investigate the molecular mechanisms of arsenic (As)-associated carcinogenesis, we performed proteomic analysis on E7 immortalized human uroepithelial cells after treatment with As in vitro. Quantitative proteomics was performed using stable isotope dimethyl labeling coupled with two-dimensional liquid chromatography peptide separation and mass spectrometry (MS)/MS analysis. Among 285 proteins, a total of 26 proteins were upregulated (ratio > 2.0) and 18 proteins were downregulated (ratio < 0.65) by As treatment, which are related to nucleotide binding, lipid metabolism, protein folding, protein biosynthesis, transcription, DNA repair, cell cycle control, and signal transduction. This study reports the potential significance of nucleophosmin (NPM) in the As-related bladder carcinogenesis. NPM was universally expressed in all of uroepithelial cell lines examined, implying that NPM may play a role in human bladder carcinogenesis. Upregulation of NPM tends to be dose- and time-dependent after As treatment. Expression of NPM was associated with cell proliferation, migration and anti-apoptosis. On the contrary, soy isoflavones inhibited the expression of NPM in vitro. The results suggest that NPM may play a role in the As-related bladder carcinogenesis, and soybean-based foods may have potential in the suppression of As/NPM-related tumorigenesis.

  20. Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

    Science.gov (United States)

    Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

    2014-11-01

    Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

  1. Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats.

    Science.gov (United States)

    Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

    2017-10-01

    Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Proteomic analysis reveals metabolic and regulatory systems involved the syntrophic and axenic lifestyle of Syntrophomonas wolfei.

    Directory of Open Access Journals (Sweden)

    Jessica Rhea Sieber

    2015-02-01

    Full Text Available Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomic approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis macromolecular stability and energy metabolism to S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

  3. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  4. Comparative proteomic and metabolomic analysis reveal the antiosteoporotic molecular mechanism of icariin from Epimedium brevicornu maxim.

    Science.gov (United States)

    Xue, Liming; Jiang, Yiping; Han, Ting; Zhang, Naidan; Qin, Luping; Xin, Hailiang; Zhang, Qiaoyan

    2016-11-04

    Icariin, a principal flavonoid glycoside of Epimedium brevicornu Maxim, has been widely proved to possess antiosteoporotic activity with promoting bone formation and decreasing bone resorption. However, the involving mechanisms remain unclear. To clear a global insight of signal pathways involved in anti-osteoporotic mechanism of icariin at proteins and metabolites level by integrating the proteomics and NMR metabonomics, in a systems biology approach. Mice were divided into sham, OVX model and icariin-treated OVX group, after 90 days treatment, difference gel electrophoresis combined with MALDI-TOF/TOF proteomics analysis on bone femur and serum metabolomics were carried out for monitor intracellular processes and elucidate anti-osteoporotic mechanism of icariin. Osteoblast and osteoclast were applied to evaluate the potential signal pathways. Twenty three proteins in bone femur, and 8 metabolites in serum, were significantly altered and identified, involving in bone remodeling, energy metabolism, cytoskeleton, lipid metabolism, MAPK signaling, Ca 2+ signaling et, al. Furthermore, animal experiment show icariin could enhance the BMD and BMC, decrease CTX-I level in ovariectomized mice. The mitochondrial membrane potential and the intracellular ATP levels were increased significantly, and the cytoskeleton were improved in icariin-treatment osteoblast and osteoclast. Icariin also increased mRNA expression of Runx2 and osterix of OB, decreased CTR and CAII mRNA expression and protein expression of P38 and JNK. However, icariin did not reveal any inhibition of the collagenolytic activity of cathepsin K, mRNA expression of MMP-9 and protein expression of ERK in osteoclast. we consider icariin as multi-targeting compounds for treating with osteoporosis, involve initiating osteoblastogenesis, inhibiting adipogenesis, and preventing osteoclast differentiation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    Science.gov (United States)

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  6. Proteomic studies of drought stress response in Fabaceae

    Directory of Open Access Journals (Sweden)

    Tanja ZADRAŽNIK

    2015-11-01

    Full Text Available Drought stress is a serious threat to crop production that influences plant growth and development and subsequently causes reduced quantity and quality of the yield. Plant stress induces changes in cell metabolism, which includes differential expression of proteins. Proteomics offer a powerful approach to analyse proteins involved in drought stress response of plants. Analyses of changes in protein abundance of legumes under drought stress are very important, as legumes play an important role in human and animal diet and are often exposed to drought. The presented results of proteomic studies of selected legumes enable better understanding of molecular mechanisms of drought stress response. The study of drought stress response of plants with proteomic approach may contribute to the development of potential drought-response markers and to the development of drought-tolerant cultivars of different legume crop species.

  7. Personalized Proteome Profiles of Healthy and Tumor Human Colon Organoids Reveal Both Individual Diversity and Basic Features of Colorectal Cancer.

    Science.gov (United States)

    Cristobal, Alba; van den Toorn, Henk W P; van de Wetering, Marc; Clevers, Hans; Heck, Albert J R; Mohammed, Shabaz

    2017-01-03

    Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A review of studies of the proteomes of circulating microparticles

    DEFF Research Database (Denmark)

    Nielsen, Christoffer Tandrup; Østergaard, Ole; Rasmussen, Niclas S

    2017-01-01

    of the specific proteins and their quantities, i.e. the proteome, in complex samples such as MPs enables an in-depth characterization of the phenotypical changes of the MPs during disease states. At present, only a limited number of proteomic studies of circulating MPs have been carried out in healthy individuals...... and disease populations. Interestingly, these studies indicate that a small set of MP-proteins, in particular, overexpression of galectin-3-binding protein (G3BP) distinguish MPs in patients with venous thromboembolism and the systemic autoimmune disease, systemic lupus erythematosus (SLE). G3BP is important...

  9. Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration.

    Directory of Open Access Journals (Sweden)

    Yuan Wang

    Full Text Available With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635.

  10. Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.).

    Science.gov (United States)

    Guo, Jinju; Wang, Peng; Cheng, Qing; Sun, Limin; Wang, Hongyu; Wang, Yutong; Kao, Lina; Li, Yanan; Qiu, Tuoyu; Yang, Wencai; Shen, Huolin

    2017-09-25

    Although cytoplasmic male sterility (CMS) is widely used for developing pepper hybrids, its molecular mechanism remains unclear. In this study, we used a high-throughput proteomics method called label-free to compare protein abundance across a pepper CMS line (A-line) and its isogenic maintainer line (B-line). Data are available via ProteomeXchange with identifier PXD006104. Approximately 324 differentially abundant protein species were identified and quantified; among which, 47 were up-accumulated and 140 were down-accumulated in the A-line; additionally, 75 and 62 protein species were specifically accumulated in the A-line and B-line, respectively. Protein species involved in pollen exine formation, pyruvate metabolic processes, the tricarboxylic acid cycle, the mitochondrial electron transport chain, and oxidative stress response were observed to be differentially accumulated between A-line and B-line, suggesting their potential roles in the regulation of pepper pollen abortion. Based on our data, we proposed a potential regulatory network for pepper CMS that unifies these processes. Artificial emasculation is a major obstacle in pepper hybrid breeding for its high labor cost and poor seed purity. While the use of cytoplasmic male sterility (CMS) in hybrid system is seriously frustrated because a long time is needed to cultivate male sterility line and its isogenic restore line. Transgenic technology is an effective and rapid method to obtain male sterility lines and its widely application has very important significance in speeding up breeding process in pepper. Although numerous studies have been conducted to select the genes related to male sterility, the molecular mechanism of cytoplasmic male sterility in pepper remains unknown. In this study, we used the high-throughput proteomic method called "label-free", coupled with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS), to perform a novel comparison of expression profiles in a CMS pepper line

  11. Proteomics

    DEFF Research Database (Denmark)

    Tølbøll, Trine Højgaard; Danscher, Anne Mette; Andersen, Pia Haubro

    2012-01-01

    to current research strategies there is a need to develop novel approaches and methods that expand understanding of the disease mechanisms involved in CHD. The objectives of the present study were to explore the potential of liquid chromatography tandem mass spectrometry (LC–MS/MS) in mapping protein...... expression in three different bovine claw tissues, and to provide a relevant functional annotation of the proteins characterized in these tissues. LC–MS/MS was used to characterize protein expression in coronary band skin (C), claw dermal (D) and lamellar (L) tissues from two heifers. A total of 388...... different proteins were identified, with 146 proteins available for identification in C, 279 proteins in D and 269 proteins in L. A functional annotation of the identified proteins was obtained using the on-line Blast2GO tool. Three hundred and sixteen of the identified proteins could be subsequently...

  12. Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis.

    Science.gov (United States)

    Guo, Zhiling; Hu, Qin; Tian, Jijing; Yan, Li; Jing, Chuanyong; Xie, Heidi Qunhui; Bao, Wenjun; Rice, Robert H; Zhao, Bin; Jiang, Guibin

    2016-11-01

    Proteomics technology is an attractive biomarker candidate discovery tool that can be applied to study large sets of biological molecules. To identify novel biomarkers and molecular targets in arsenic-induced skin lesions, we have determined the protein profile of arsenic-affected human epidermal stratum corneum by shotgun proteomics. Samples of palm and foot sole from healthy subjects were analyzed, demonstrating similar protein patterns in palm and sole. Samples were collected from the palms of subjects with arsenic keratosis (lesional and adjacent non-lesional samples) and arsenic-exposed subjects without lesions (normal). Samples from non-exposed healthy individuals served as controls. We found that three proteins in arsenic-exposed lesional epidermis were consistently distinguishably expressed from the unaffected epidermis. One of these proteins, the cadherin-like transmembrane glycoprotein, desmoglein 1 (DSG1) was suppressed. Down-regulation of DSG1 may lead to reduced cell-cell adhesion, resulting in abnormal epidermal differentiation. The expression of keratin 6c (KRT6C) and fatty acid binding protein 5 (FABP5) were significantly increased. FABP5 is an intracellular lipid chaperone that plays an essential role in fatty acid metabolism in human skin. This raises a possibility that overexpression of FABP5 may affect the proliferation or differentiation of keratinocytes by altering lipid metabolism. KRT6C is a constituent of the cytoskeleton that maintains epidermal integrity and cohesion. Abnormal expression of KRT6C may affect its structural role in the epidermis. Our findings suggest an important approach for future studies of arsenic-mediated toxicity and skin cancer, where certain proteins may represent useful biomarkers of early diagnoses in high-risk populations and hopefully new treatment targets. Further studies are required to understand the biological role of these markers in skin pathogenesis from arsenic exposure. Copyright © 2016 Elsevier Ltd

  13. Proteomic analysis revealed alterations of the Plasmodium falciparum metabolism following salicylhydroxamic acid exposure

    Directory of Open Access Journals (Sweden)

    Torrentino-Madamet M

    2011-09-01

    Full Text Available Marylin Torrentino-Madamet1, Lionel Almeras2, Christelle Travaillé1, Véronique Sinou1, Matthieu Pophillat3, Maya Belghazi4, Patrick Fourquet3, Yves Jammes5, Daniel Parzy11UMR-MD3, Université de la Méditerranée, Antenne IRBA de Marseille (IMTSSA, Le Pharo, 2Unité de Recherche en Biologie et Epidémiologie Parasitaires, Antenne IRBA de Marseille (IMTSSA, Le Pharo, 3Centre d'Immunologie de Marseille Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de la Méditerranée, 4Centre d'Analyse Protéomique de Marseille, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, 5UMR-MD2, Physiologie et Physiopathologie en Conditions d'Oxygénations Extrêmes, Institut Fédératif de Recherche Jean Roche, Faculté de Médecine Nord, Marseille, FranceObjectives: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a “branched respiratory chain.” This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM, showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure.Design: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed.Results: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins

  14. Proteomics analysis of human skeletal muscle reveals novel abnormalities in obesity and type 2 diabetes

    DEFF Research Database (Denmark)

    Hwang, Hyonson; Bowen, Benjamin P; Lefort, Natalie

    2010-01-01

    changes involving the use of proteomics was used here. RESEARCH DESIGN AND METHODS: Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry......OBJECTIVE : Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new......-based quantification using normalized spectral abundance factors. RESULTS: Of 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P

  15. Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

    KAUST Repository

    Zhang, Huoming; Zhao, Changqing; Li, Xin; Zhu, Yi; Gan, Chee Sian; Wang, Yong; Ravasi, Timothy; Qian, Pei-Yuan; Wong, Siew Cheng; Sze, Siu Kwan

    2010-01-01

    Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

  16. Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

    KAUST Repository

    Zhang, Huoming

    2010-07-02

    Human monocytes\\' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

  17. Proteomics Coupled with Metabolite and Cell Wall Profiling Reveal Metabolic Processes of a Developing Rice Stem Internode

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Williams, Brad J.; Thangella, Padmavathi A. V.; Ladak, Adam; Schepmoes, Athena A.; Olivos, Hernando J.; Zhao, Kangmei; Callister, Stephen J.; Bartley, Laura E.

    2017-07-13

    Internodes of grass stems function in mechanical support, transport, and, in some species, are a major sink organ for carbon in the form of cell wall polymers. This study reports cell wall composition, proteomic and metabolite analyses of the rice elongating internode. Along eight segments of the second rice internode (internode II) at booting stage, cellulose, lignin, and xylose increase as a percentage of cell wall material from the younger to the older internode segments, indicating active cell wall synthesis. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) of trypsin-digested peptides of size-fractionated proteins extracted from this internode at booting reveals 2547proteins with at least two unique peptides. The dataset includes many glycosyltransferases, acyltransferases, glycosyl hydrolases, cell wall-localized proteins, and protein kinases that have or may have functions in cell wall biosynthesis or remodeling. Phospho-enrichment of the internode II peptides identified 21 unique phosphopeptides belonging to 20 phosphoproteins including an LRR-III family receptor like kinase. GO over-representation and KEGG pathway analyses highlight the abundances of internode proteins involved in biosynthetic processes, especially the synthesis of secondary metabolites such as phenylpropanoids and flavonoids. LC-MS of hot methanol-extracted secondary metabolites from internode II at four stages (elongation, early mature, mature and post mature) indicates that secondary metabolites in stems are distinct from those of roots and leaves, and differ during stem maturation. This work fills a void of knowledge of proteomics and metabolomics data for grass stems, specifically for rice, and provides baseline knowledge for more detailed studies of cell wall synthesis and other biological processes during internode development, toward improving grass agronomic properties.

  18. Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

    Science.gov (United States)

    Yang, Xiaoli; Li, Hongtao; Zhang, Chengdong; Lin, Zhidi; Zhang, Xinhua; Zhang, Youjie; Yu, Yanbao; Liu, Kun; Li, Muyan; Zhang, Yuening; Lv, Wenxin; Xie, Yuanliang; Lu, Zheng; Wu, Chunlei; Teng, Ruobing; Lu, Shaoming; He, Min; Mo, Zengnan

    2015-10-01

    Prostatitis is one of the most common urological problems afflicting adult men. The etiology and pathogenesis of nonbacterial prostatitis, which accounts for 90-95% of cases, is largely unknown. As serum proteins often indicate the overall pathologic status of patients, we hypothesized that protein biomarkers of prostatitis might be identified by comparing the serum proteomes of patients with and without nonbacterial prostatitis. All untreated samples were collected from subjects attending the Fangchenggang Area Male Health and Examination Survey (FAMHES). We profiled pooled serum samples from four carefully selected groups of patients (n = 10/group) representing the various categories of nonbacterial prostatitis (IIIa, IIIb, and IV) and matched healthy controls using a mass spectrometry-based 4-plex iTRAQ proteomic approach. More than 160 samples were validated by ELISA. Overall, 69 proteins were identified. Among them, 42, 52, and 37 proteins were identified with differential expression in Category IIIa, IIIb, and IV prostatitis, respectively. The 19 common proteins were related to immunity and defense, ion binding, transport, and proteolysis. Two zinc-binding proteins, superoxide dismutase 3 (SOD3), and carbonic anhydrase I (CA1), were significantly higher in all types of prostatitis than in the control. A receiver operating characteristic curve estimated sensitivities of 50.4 and 68.1% and specificities of 92.1 and 83.8% for CA1 and SOD3, respectively, in detecting nonbacterial prostatitis. The serum CA1 concentration was inversely correlated to the zinc concentration in expressed-prostatic secretions. Our findings suggest that SOD3 and CA1 are potential diagnostic markers of nonbacterial prostatitis, although further large-scale studies are required. The molecular profiles of nonbacterial prostatitis pathogenesis may lay a foundation for discovery of new therapies. © 2015 Wiley Periodicals, Inc.

  19. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    Science.gov (United States)

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  20. Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization.

    Science.gov (United States)

    Müller, Daniel B; Schubert, Olga T; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A

    2016-10-01

    Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization*

    Science.gov (United States)

    Müller, Daniel B.; Schubert, Olga T.; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A.

    2016-01-01

    Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. PMID:27457762

  2. Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

    Science.gov (United States)

    Naboulsi, Wael; Megger, Dominik A; Bracht, Thilo; Kohl, Michael; Turewicz, Michael; Eisenacher, Martin; Voss, Don Marvin; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

    2016-01-04

    Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

  3. Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

    Directory of Open Access Journals (Sweden)

    Marangoni Sérgio

    2009-04-01

    Full Text Available Abstract Background Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area – BA22p identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6 and glial fibrillary acidic protein (GFAP were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

  4. The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features

    Directory of Open Access Journals (Sweden)

    Carvalho Carla M

    2012-01-01

    Full Text Available Abstract Background Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies. Methods vB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry. Results and conclusions The DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and ϕ29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle. Conclusions Genes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS degradation, which has not been reported

  5. Differential proteomic analysis reveals sequential heat stress-responsive regulatory network in radish (Raphanus sativus L.) taproot.

    Science.gov (United States)

    Wang, Ronghua; Mei, Yi; Xu, Liang; Zhu, Xianwen; Wang, Yan; Guo, Jun; Liu, Liwang

    2018-05-01

    Differential abundance protein species (DAPS) involved in reducing damage and enhancing thermotolerance in radish were firstly identified. Proteomic analysis and omics association analysis revealed a HS-responsive regulatory network in radish. Heat stress (HS) is a major destructive factor influencing radish production and supply in summer, for radish is a cool season vegetable crop being susceptible to high temperature. In this study, the proteome changes of radish taproots under 40 °C treatment at 0 h (Control), 12 h (Heat12) and 24 h (Heat24) were analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) approach. In total, 2258 DAPS representing 1542 differentially accumulated uniprotein species which respond to HS were identified. A total of 604, 910 and 744 DAPS was detected in comparison of Control vs. Heat12, Control vs. Heat24, and Heat12 vs. Heat24, respectively. Gene ontology and pathway analysis showed that annexin, ubiquitin-conjugating enzyme, ATP synthase, heat shock protein (HSP) and other stress-related proteins were predominately enriched in signal transduction, stress and defense pathways, photosynthesis and energy metabolic pathways, working cooperatively to reduce stress-induced damage in radish. Based on iTRAQ combined with the transcriptomics analysis, a schematic model of a sequential HS-responsive regulatory network was proposed. The initial sensing of HS occurred at the plasma membrane, and then key components of stress signal transduction triggered heat-responsive genes in the plant protective metabolism to re-establish homeostasis and enhance thermotolerance. These results provide new insights into characteristics of HS-responsive DAPS and facilitate dissecting the molecular mechanisms underlying heat tolerance in radish and other root crops.

  6. Application of Proteomics to the Study of Pollination Drops

    Directory of Open Access Journals (Sweden)

    Natalie Prior

    2013-04-01

    Full Text Available Premise of the study: Pollination drops are a formative component in gymnosperm pollen-ovule interactions. Proteomics offers a direct method for the discovery of proteins associated with this early stage of sexual reproduction. Methods: Pollination drops were sampled from eight gymnosperm species: Chamaecyparis lawsoniana (Port Orford cedar, Ephedra monosperma, Ginkgo biloba, Juniperus oxycedrus (prickly juniper, Larix ×marschlinsii, Pseudotsuga menziesii (Douglas-fir, Taxus ×media, and Welwitschia mirabilis. Drops were collected by micropipette using techniques focused on preventing sample contamination. Drop proteins were separated using both gel and gel-free methods. Tandem mass spectrometric methods were used including a triple quadrupole and an Orbitrap. Results: Proteins are present in all pollination drops. Consistency in the protein complement over time was shown in L. ×marschlinsii. Representative mass spectra from W. mirabilis chitinase peptide and E. monosperma serine carboxypeptidase peptide demonstrated high quality results. We provide a summary of gymnosperm pollination drop proteins that have been discovered to date via proteomics. Discussion: Using proteomic methods, a dozen classes of proteins have been identified to date. Proteomics presents a way forward in deepening our understanding of the biological function of pollination drops.

  7. Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

    Science.gov (United States)

    Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

    2015-02-01

    Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

  8. Comparative proteomic analyses reveal the proteome response to short-term drought in Italian ryegrass (Lolium multiflorum.

    Directory of Open Access Journals (Sweden)

    Ling Pan

    Full Text Available Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport.

  9. Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

    OpenAIRE

    Manzanares-Miralles, Lara; Bayram, Ozgur; Sarikaya-Bayram, Ozlem; Smith, Elizabeth B.; Dolan, Stephen K.; Jones, Gary W.; Doyle, Sean

    2016-01-01

    Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus,which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p = 0.0018) ...

  10. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  11. Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger.

    Science.gov (United States)

    Manzanares-Miralles, Lara; Sarikaya-Bayram, Özlem; Smith, Elizabeth B; Dolan, Stephen K; Bayram, Özgür; Jones, Gary W; Doyle, Sean

    2016-01-10

    Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus, which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p=0.0018) required for homocysteine generation from S-adenosylhomocysteine (SAH), and spermidine synthase (p=0.0068), involved in the recycling of Met, was observed. Analysis of Met-related metabolites revealed significant increases in the levels of Met and adenosine, in correlation with proteomic data. Methyltransferase MT-II is responsible for bisthiobis(methylthio)gliotoxin (BmGT) formation, deletion of MT-II abolished BmGT formation and led to increased GT sensitivity in A. niger. Proteomic analysis also revealed that GT exposure also significantly (pniger. Thus, it provides new opportunities to exploit the response of GT-naïve fungi to GT. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

    Energy Technology Data Exchange (ETDEWEB)

    Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.; Gritsenko, Marina A.; Jacobs, Jon M.; Smith, Richard D.

    2014-04-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  13. Differential proteomics reveals novel insights into Nosema-honey bee interactions.

    Science.gov (United States)

    Kurze, Christoph; Dosselli, Ryan; Grassl, Julia; Le Conte, Yves; Kryger, Per; Baer, Boris; Moritz, Robin F A

    2016-12-01

    Host manipulation is a common strategy by parasites to reduce host defense responses, enhance development, host exploitation, reproduction and, ultimately, transmission success. As these parasitic modifications can reduce host fitness, increased selection pressure may result in reciprocal adaptations of the host. Whereas the majority of studies on host manipulation have explored resistance against parasites (i.e. ability to prevent or limit an infection), data describing tolerance mechanisms (i.e. ability to limit harm of an infection) are scarce. By comparing differential protein abundance, we provide evidence of host-parasite interactions in the midgut proteomes of N. ceranae-infected and uninfected honey bees from both Nosema-tolerant and Nosema-sensitive lineages. We identified 16 proteins out of 661 protein spots that were differentially abundant between experimental groups. In general, infections of Nosema resulted in an up-regulation of the bee's energy metabolism. Additionally, we identified 8 proteins that were differentially abundant between tolerant and sensitive honey bees regardless of the Nosema infection. Those proteins were linked to metabolism, response to oxidative stress and apoptosis. In addition to bee proteins, we also identified 3 Nosema ceranae proteins. Interestingly, abundance of two of these Nosema proteins were significantly higher in infected Nosema-sensitive honeybees relative to the infected Nosema-tolerant lineage. This may provide a novel candidate for studying the molecular interplay between N. ceranae and its honey bee host in more detail. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Revealing proteins associated with symbiotic germination of Gastrodia elata by proteomic analysis.

    Science.gov (United States)

    Zeng, Xu; Li, Yuanyuan; Ling, Hong; Chen, Juan; Guo, Shunxing

    2018-03-06

    Gastrodia elata, a mycoheterotrophic orchid, is a well-known medicinal herb. In nature, the seed germination of G. elata requires proper fungal association, because of the absence of endosperm. To germinate successfully, G. elata obtains nutrition from mycorrhizal fungi such as Mycena. However, Mycena is not able to supply nutrition for the further development and enlargement of protocorms into tubers, flowering and fruit setting of G. elata. To date, current genomic studies on this topic are limited. Here we used the proteomic approach to explore changes in G. elata at different stages of symbiotic germination. Using mass spectrometry, 3787 unique proteins were identified, of which 599 were classified as differentially accumulated proteins. Most of these differentially accumulated proteins were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Among them, the defense genes (e.g., pathogenesis-/wound-related proteins, peroxidases, and serine/threonine-protein kinase) were highly expressed in late-stage protocorms, suggesting that fungal colonization triggered the significant defense responses of G. elata. The present study indicated the metabolic change and defensive reaction could disrupt the balance between Mycena and G. elata during mycorrhizal symbiotic germination.

  15. Quantitative proteome profiling of human myoma and myometrium tissue reveals kinase expression signatures with potential for therapeutic intervention

    NARCIS (Netherlands)

    Lemeer, Simone; Gholami, Amin Moghaddas; Wu, Zhixiang; Kuster, Bernhard

    2015-01-01

    Uterine leiomyomas are benign tumors affecting a large proportion of the female population. Despite the very high prevalence, the molecular basis for understanding the onset and development of the disease are still poorly understood. In this study, we profiled the proteomes and kinomes of leiomyoma

  16. Quantitative proteomics revealed partial fungistatic mechanism of ammonia against conidial germination of nematode-trapping fungus Arthrobotrys oligospora ATCC24927.

    Science.gov (United States)

    Liu, Tong; Tian, Dong-Wei; Zou, Li-Juan; Liu, Fang-Yu; Can, Qi-Yan; Yang, Jin-Kui; Xu, Jian-Ping; Huang, Xiao-Wei; Xi, Jia-Qin; Zhu, Ming-Liang; Mo, Ming-He; Zhang, Ke-Qin

    2018-05-01

    Ammonia is one of the fungistatic factors in soil that can suppress conidial germination, but the molecular mechanism underlying the suppression is unknown. In this study, the proteomes of fungistatic conidia, fresh conidia and germinated conidia of Arthrobotrys oligospora ATCC24927 were determined and quantified. The protein expression profile of fungistatic conidia was significantly different from those in the other two conditions. 281 proteins were down expressed in fungistatic conidia and characterized by GO annotation. Gene transcription analysis and inhibition of puromycin (a protein translation inhibitor) on conidial germination suggested that down expression of 33 protein translation related proteins might well result in repression of protein synthesis and inhibition of conidial germination. In addition, 16 down-expressed proteins were mapped to the Ras/mitogen-activated protein (Ras/MAP) regulatory networks which regulate conidial DNA synthesis. The conidial DNA synthesis was found to be definitely inhibited under by ammonia, and function studies of two Ras/MAP proteins by using knock-out strains provided partial evidence that Ras/MAP pathway regulate the conidial germination. These results suggested that down-expression of Ras/MAP related proteins might result in inhibition of DNA synthesis and finally result in inhibition conidial germination. This study revealed partial fungistatic mechanism of ammonia against conidial germination. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

    Directory of Open Access Journals (Sweden)

    Susann Kummer

    Full Text Available We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1 infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC. We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

  18. Proteome analysis reveals an energy-dependent central process for Populus×canadensis seed germination.

    Science.gov (United States)

    Zhang, Hong; Zhou, Ke-Xin; Wang, Wei-Qing; Liu, Shu-Jun; Song, Song-Quan

    2017-06-01

    Poplar (Populus×canadensis) seeds rapidly germinated in darkness at 10, 15, and 20°C and reached 50% seed germination after about 22, 4.5, and 3.5h, respectively. Germination of poplar seeds was markedly inhibited by abscisic acid (ABA) at 50μM and cycloheximide (CHX) at 100μM, and these inhibitive roles were temperature-dependent. In the present study, mature poplar seeds were used to investigate the differentially changed proteome of seeds germinating in water, ABA, and CHX. A total of 130 protein spots showed a significant change (1.5-fold increase/decrease, Pgermination of poplar seeds is closely related with the increase in those proteins involved in amino acid and lipid metabolism, the tricarboxylic acid cycle and pentose phosphate pathway, protein synthesis and destination, cell defense and rescue, and degradation of storage proteins. ABA and CHX inhibit the germination of poplar seeds by decreasing the protein abundance associated with protein proteolysis, protein folding, and storage proteins. We conclude that poplar seed germination is an energy-dependent active process, and is accompanied by increasing amino acid activation, protein synthesis and destination, as well as cell defense and rescue, and degradation of storage proteins. Copyright © 2017 Elsevier GmbH. All rights reserved.

  19. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    Science.gov (United States)

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  20. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

    Science.gov (United States)

    Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

    2013-11-19

    JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

  1. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  2. A review of studies of the proteomes of circulating microparticles

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T.; Østergaard, Ole; Rasmussen, Niclas S.

    2017-01-01

    understood, MVs are involved in trafficking of information from cell-to-cell, and are implicated in the regulation of immunity, thrombosis, and coagulation. Different subtypes of extracellular MVs exist. This review focuses on the cell membrane-derived shedded MVs (ranging in size from 200 to 1000 nm...... conditions such as cancer and chronic viral infections. This review highlights the methodology and results of the proteome studies behind these discoveries and places them in a pathophysiological and biomarker perspective....

  3. Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism.

    Science.gov (United States)

    Houston, Norma L; Hajduch, Martin; Thelen, Jay J

    2009-10-01

    Seed maturation or seed filling is a phase of development that plays a major role in the storage reserve composition of a seed. In many plant seeds photosynthesis plays a major role in this process, although oilseeds, such as castor (Ricinus communis), are capable of accumulating oil without the benefit of photophosphorylation to augment energy demands. To characterize seed filling in castor, a systematic quantitative proteomics study was performed. Two-dimensional gel electrophoresis was used to resolve and quantify Cy-dye-labeled proteins expressed at 2, 3, 4, 5, and 6 weeks after flowering in biological triplicate. Expression profiles for 660 protein spot groups were established, and of these, 522 proteins were confidently identified by liquid chromatography-tandem mass spectrometry by mining against the castor genome. Identified proteins were classified according to function, and the most abundant groups of proteins were involved in protein destination and storage (34%), energy (19%), and metabolism (15%). Carbon assimilatory pathways in castor were compared with previous studies of photosynthetic oilseeds, soybean (Glycine max) and rapeseed (Brassica napus). These comparisons revealed differences in abundance and number of protein isoforms at numerous steps in glycolysis. One such difference was the number of enolase isoforms and their sum abundance; castor had approximately six times as many isoforms as soy and rapeseed. Furthermore, Rubisco was 11-fold less prominent in castor compared to rapeseed. These and other differences suggest some aspects of carbon flow, carbon recapture, as well as ATP and NADPH production in castor differs from photosynthetic oilseeds.

  4. Toxicological effects of benzo(a)pyrene, DDT and their mixture on the green mussel Perna viridis revealed by proteomic and metabolomic approaches.

    Science.gov (United States)

    Song, Qinqin; Chen, Hao; Li, Yuhu; Zhou, Hailong; Han, Qian; Diao, Xiaoping

    2016-02-01

    Benzo(a)pyrene (BaP) and dichlorodiphenyltrichloroethane (DDT) are persistent organic pollutants and environmental estrogens (EEs) with known toxicity towards the green mussel, Perna viridis. In this study, the toxic effects of BaP (10 µg/L) and DDT (10 µg/L) and their mixture were assessed in green mussel gills with proteomic and metabolomic approaches. Metabolic responses indicated that BaP mainly caused disturbance in osmotic regulation by significantly decrease in branched chain amino acids, dimethylamine and dimethylglycine in gills of male green mussels after exposure for 7 days. DDT mainly caused disturbance in osmotic regulation and energy metabolism by differential alteration of betaine, dimethylamine, dimethylglycine, amino acids, and succinate in gills of male green mussels. However, the mixture of BaP and DDT didn't show obvious metabolite changes. Proteomic analysis showed different protein expression profiles between different treatment groups, which demonstrated that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced cell apoptosis, disturbance in protein digestion and energy metabolism in gills of green mussels, whereas DDT exposure altered proteins that were associated with oxidative stress, cytoskeleton and cell structure, protein digestion and energy metabolism. However, the mixture of BaP and DDT affected proteins related to the oxidative stress, cytoskeleton and cell structure, protein biosynthesis and modification, energy metabolism, growth and apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Contribution of proteomics to the study of plant pathogenic fungi.

    Science.gov (United States)

    Gonzalez-Fernandez, Raquel; Jorrin-Novo, Jesus V

    2012-01-01

    Phytopathogenic fungi are one of the most damaging plant parasitic organisms, and can cause serious diseases and important yield losses in crops. The study of the biology of these microorganisms and the interaction with their hosts has experienced great advances in recent years due to the development of moderm, holistic and high-throughput -omic techniques, together with the increasing number of genome sequencing projects and the development of mutants and reverse genetics tools. We highlight among these -omic techniques the importance of proteomics, which has become a relevant tool in plant-fungus pathosystem research. Proteomics intends to identify gene products with a key role in pathogenicity and virulence. These studies would help in the search of key protein targets and in the development of agrochemicals, which may open new ways for crop disease diagnosis and protection. In this review, we made an overview on the contribution of proteomics to the knowledge of life cycle, infection mechanisms, and virulence of the plant pathogenic fungi. Data from current, innovative literature, according to both methodological and experimental systems, were summarized and discussed. Specific sections were devoted to the most studied fungal phytopathogens: Botrytis cinerea, Sclerotinia sclerotiorum, and Fusarium graminearum.

  6. Study on the effect of irradiation on algae by proteomics

    International Nuclear Information System (INIS)

    Choi, Jong Il; Yoon, Yo Han; Kim, Jae Hun

    2010-06-01

    Algae has been utilized as food material from long time ago, and recently newly recognized as functional materials and the source of bio-fuel. But, the study on the algae is just beginning and the study on protein expression and growth by the change of condition was not reported. In this study, the effect of radiation on the protein expression was investigated for the protection mechanisms and new genome source and furthermore, isolation of new mutant strains. To monitor the growth of algae, absorbance and FDA staining methods were developed and the content of lipid of algae species were measured. With these methods, the radiation sensitivity of algae species was determined. To investigate the proteome of algae, 2D-electrophoresis methods was applied. From the comparison of proteomes, the radiation specific expressed protein was identified as thioredoxin-h and its nucleotide sequences was defined. The expression of thioredoxin-h was further defined on the mRNA level. Also, the extract of algae species was analyzed for its antioxidant activity and polyphenolic content. The changes in antioxidant activity of extract by radiation was investigated. From the radiation experiments, mutant Spirogyra species having higher resistant against radical stress was obtained. The mutant strain has higher antioxidant activity. This results can provide the proteome date and mutation technology of algae and further contribute in the activation of fishery industry and national health enhancement

  7. Proteomic analysis reveals resistance mechanism against biofuel hexane in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Liu Jie

    2012-09-01

    Full Text Available Abstract Background Recent studies have demonstrated that photosynthetic cyanobacteria could be an excellent cell factory to produce renewable biofuels and chemicals due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources. Biosynthesis of carbon-neutral biofuel alkanes with good chemical and physical properties has been proposed. However, to make the process economically feasible, one major hurdle to improve the low cell tolerance to alkanes needed to be overcome. Results Towards the goal to develop robust and high-alkane-tolerant hosts, in this study, the responses of model cyanobacterial Synechocystis PCC 6803 to hexane, a representative of alkane, were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. In total, 1,492 unique proteins were identified, representing about 42% of all predicted protein in the Synechocystis genome. Among all proteins identified, a total of 164 and 77 proteins were found up- and down-regulated, respectively. Functional annotation and KEGG pathway enrichment analyses showed that common stress responses were induced by hexane in Synechocystis. Notably, a large number of transporters and membrane-bound proteins, proteins against oxidative stress and proteins related to sulfur relay system and photosynthesis were induced, suggesting that they are possibly the major protection mechanisms against hexane toxicity. Conclusion The study provided the first comprehensive view of the complicated molecular mechanism employed by cyanobacterial model species, Synechocystis to defend against hexane stress. The study also provided a list of potential targets to engineer Synechocystis against hexane stress.

  8. Proteomic study on gender differences in aging kidney of mice

    Directory of Open Access Journals (Sweden)

    Cristobal Susana

    2009-04-01

    Full Text Available Abstract Background This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS. Results This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes. Conclusion Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.

  9. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

    Science.gov (United States)

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

    2014-07-01

    Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  10. Environmental proteomics reveals early microbial community responses to biostimulation at a uranium- and nitrate-contaminated site

    Energy Technology Data Exchange (ETDEWEB)

    Chourey, Karuna [ORNL; Nissen, Silke [ORNL; Vishnivetskaya, T. [University of Tennessee, Knoxville (UTK); Shah, Manesh B [ORNL; Pffifner, Susan [University of Tennessee, Knoxville (UTK); Hettich, Robert {Bob} L [ORNL; Loeffler, Frank E [ORNL

    2013-01-01

    High performance mass spectrometry instrumentation coupled with improved protein extraction techniques enable metaproteomics to identify active members of soil and groundwater microbial communities. Metaproteomics workflows were applied to study the initial responses (i.e., 4 days post treatment) of the indigenous aquifer microbiota to biostimulation with emulsified vegetable oil (EVO) at a uranium-contaminated site. Members of the Betaproteobacteria (i.e., Dechloromonas, Ralstonia, Rhodoferax, Polaromonas, Delftia, Chromobacterium) and Firmicutes dominated the biostimulated aquifer community. Proteome characterization revealed distinct differences in protein expression between the microbial biomass collected from groundwater influenced by biostimulation and groundwater collected up-gradient of the EVO injection points. In particular, proteins involved in ammonium assimilation, EVO degradation, and polyhydroxybutyrate (PHB) granule formation were prominent following biostimulation. Interestingly, the atypical NosZ of a Dechloromonas sp. was highly expressed suggesting active nitrous oxide (N2O) respiration. c-type cytochromes were barely detected, as was citrate synthase, a biomarker for hexavalent uranium reduction activity, suggesting that metal reduction has not commenced 4 days post EVO delivery. Environmental metaproteomics identified microbial community responses to biostimulation and elucidated active pathways demonstrating the value of this technique for complementing nucleic acid-based approaches.

  11. Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

    Science.gov (United States)

    Blundon, Malachi A; Schlesinger, Danielle R; Parthasarathy, Amritha; Smith, Samantha L; Kolev, Hannah M; Vinson, David A; Kunttas-Tatli, Ezgi; McCartney, Brooke M; Minden, Jonathan S

    2016-07-15

    Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development. © 2016. Published by The Company of Biologists Ltd.

  12. Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

    KAUST Repository

    Dineshram, R.; Q., Quan; Sharma, Rakesh; Chandramouli, Kondethimmanahalli; Yalamanchili, Hari Krishna; Chu, Ivan; Thiyagarajan, Vengatesen

    2015-01-01

    © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

  13. Proteomic Analysis of Mouse Oocytes Reveals 28 Candidate Factors of the "Reprogrammome"

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Siatkowski, M.; Paudel, Y.; Balbach, S.T.; Baeumer, N.; Crosetto, N.; Drexler, H.C.A.; Fuellen, G.; Boiani, M.

    2011-01-01

    The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date,

  14. Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

    KAUST Repository

    Dineshram, R.

    2015-10-28

    © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world\\'s edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

  15. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism

    DEFF Research Database (Denmark)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill

    2015-01-01

    Elucidating how and to what extent lipid metabolism is remodeled under changing conditions is essential for understanding cellular physiology. Here, we analyzed proteome and lipidome dynamics to investigate how regulation of lipid metabolism at the global scale supports remodeling of cellular...

  16. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  17. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics

    NARCIS (Netherlands)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic Y; Jennings, Paul

    2015-01-01

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to

  18. A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles

    DEFF Research Database (Denmark)

    Wagner, Sebastian Alexander; Beli, Petra; Weinert, Brian Tate

    2011-01-01

    Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single...

  19. Strain-resolved microbial community proteomics reveals simultaneous aerobic and anaerobic function during gastrointestinal tract colonization of a preterm infant

    Directory of Open Access Journals (Sweden)

    Brandon eBrooks

    2015-07-01

    Full Text Available While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13-21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains of Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.

  20. iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis.

    Science.gov (United States)

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

    2016-01-01

    Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of

  1. Proteome Analysis Reveals Extensive Light Stress-Response Reprogramming in the Seagrass Zostera muelleri (Alismatales, Zosteraceae) Metabolism.

    Science.gov (United States)

    Kumar, Manoj; Padula, Matthew P; Davey, Peter; Pernice, Mathieu; Jiang, Zhijian; Sablok, Gaurav; Contreras-Porcia, Loretto; Ralph, Peter J

    2016-01-01

    Seagrasses are marine ecosystem engineers that are currently declining in abundance at an alarming rate due to both natural and anthropogenic disturbances in ecological niches. Despite reports on the morphological and physiological adaptations of seagrasses to extreme environments, little is known of the molecular mechanisms underlying photo-acclimation, and/or tolerance in these marine plants. This study applies the two-dimensional isoelectric focusing (2D-IEF) proteomics approach to identify photo-acclimation/tolerance proteins in the marine seagrass Zostera muelleri . For this, Z. muelleri was exposed for 10 days in laboratory mesocosms to saturating (control, 200 μmol photons m -2 s -1 ), super-saturating (SSL, 600 μmol photons m -2 s -1 ), and limited light (LL, 20 μmol photons m -2 s -1 ) irradiance conditions. Using LC-MS/MS analysis, 93 and 40 protein spots were differentially regulated under SSL and LL conditions, respectively, when compared to the control. In contrast to the LL condition, Z. muelleri robustly tolerated super-saturation light than control conditions, evidenced by their higher relative maximum electron transport rate and minimum saturating irradiance values. Proteomic analyses revealed up-regulation and/or appearances of proteins belonging to the Calvin-Benson and Krebs cycle, glycolysis, the glycine cleavage system of photorespiration, and the antioxidant system. These proteins, together with those from the inter-connected glutamate-proline-GABA pathway, shaped Z. muelleri photosynthesis and growth under SSL conditions. In contrast, the LL condition negatively impacted the metabolic activities of Z. muelleri by down-regulating key metabolic enzymes for photosynthesis and the metabolism of carbohydrates and amino acids, which is consistent with the observation with lower photosynthetic performance under LL condition. This study provides novel insights into the underlying molecular photo-acclimation mechanisms in Z. muelleri , in addition

  2. Proteomic Studies on Human and Experimental Cerebral Malaria

    KAUST Repository

    Moussa, Ehab

    2012-07-01

    Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially

  3. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.

  4. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highlydifferentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram

  5. iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

    Science.gov (United States)

    Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

    2016-01-15

    Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

  6. Differential proteomics study of platelets in asymptomatic constitutional macrothrombocytopenia: altered levels of cytoskeletal proteins.

    Science.gov (United States)

    Karmakar, Shilpita; Saha, Sutapa; Banerjee, Debasis; Chakrabarti, Abhijit

    2015-01-01

    Harris platelet syndrome (HPS), also known as asymptomatic constitutional macrothrombocytopenia (ACMT), is an autosomal dominant platelet disorder characterized by mild-to-severe thrombocytopenia and giant platelets with normal platelet aggregation and absence of bleeding symptoms. We have attempted a comparative proteomics study for profiling of platelet proteins in healthy vs. pathological states to discover characteristic protein expression changes in macrothrombocytes and decipher the factors responsible for the functionally active yet morphologically distinct platelets. We have used 2-D gel-based protein separation techniques coupled with MALDI-ToF/ToF-based mass spectrometric identification and characterization of the proteins to investigate the differential proteome profiling of platelet proteins isolated from the peripheral blood samples of patients and normal volunteers. Our study revealed altered levels of actin-binding proteins such as myosin light chain, coactosin-like protein, actin-related protein 2/3 complex, and transgelin2 that hint toward the cytoskeletal changes necessary to maintain the structural and functional integrity of macrothrombocytes. We have also observed over expressed levels of peroxiredoxin2 that signifies the prevailing oxidative stress in these cells. Additionally, altered levels of protein disulfide isomerase and transthyretin provide insights into the measures adapted by the macrothrombocytes to maintain their normal functional activity. This first proteomics study of platelets from ACMT may provide an understanding of the structural stability and normal functioning of these platelets in spite of their large size. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Papetti, Moreno; Rigbolt, Kristoffer T G

    2016-01-01

    , we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We...... identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF......-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling....

  8. The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions

    OpenAIRE

    Vazquez-Gutierrez, Pamela; Stevens, Marc J. A.; Gehrig, Peter; Barkow-Oesterreicher, Simon; Lacroix, Christophe; Chassard, Christophe

    2017-01-01

    Background Bifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-seq...

  9. Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

    Science.gov (United States)

    Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

    2016-12-01

    During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

  10. Salinity-induced inhibition of growth in the aquatic pteridophyte Azolla microphylla primarily involves inhibition of photosynthetic components and signaling molecules as revealed by proteome analysis.

    Science.gov (United States)

    Thagela, Preeti; Yadav, Ravindra Kumar; Mishra, Vagish; Dahuja, Anil; Ahmad, Altaf; Singh, Pawan Kumar; Tiwari, Budhi Sagar; Abraham, Gerard

    2017-01-01

    Salinity stress causes adverse physiological and biochemical changes in the growth and productivity of a plant. Azolla, a symbiotic pteridophyte and potent candidate for biofertilizer due to its nitrogen fixation ability, shows reduced growth and nitrogen fixation during saline stress. To better understand regulatory components involved in salinity-induced physiological changes, in the present study, Azolla microphylla plants were exposed to NaCl (6.74 and 8.61 ds/m) and growth, photochemical reactions of photosynthesis, ion accumulation, and changes in cellular proteome were studied. Maximum dry weight was accumulated in control and untreated plant while a substantial decrease in dry weight was observed in the plants exposed to salinity. Exposure of the organism to different concentrations of salt in hydroponic conditions resulted in differential level of Na + and K + ion accumulation. Comparative analysis of salinity-induced proteome changes in A. microphylla revealed 58 salt responsive proteins which were differentially expressed during the salt exposure. Moreover, 42 % spots among differentially expressed proteins were involved in different signaling events. The identified proteins are involved in photosynthesis, energy metabolism, amino acid biosynthesis, protein synthesis, and defense. Downregulation of these key metabolic proteins appears to inhibit the growth of A. microphylla in response to salinity. Altogether, the study revealed that in Azolla, increased salinity primarily affected signaling and photosynthesis that in turn leads to reduced biomass.

  11. Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

    Science.gov (United States)

    Grabek, Katharine R.; Karimpour-Fard, Anis; Epperson, L. Elaine; Hindle, Allyson; Hunter, Lawrence E.

    2011-01-01

    The hibernator's heart functions continuously and avoids damage across the wide temperature range of winter heterothermy. To define the molecular basis of this phenotype, we quantified proteomic changes in the 13-lined ground squirrel heart among eight distinct physiological states encompassing the hibernator's year. Unsupervised clustering revealed a prominent seasonal separation between the summer homeotherms and winter heterotherms, whereas within-season state separation was limited. Further, animals torpid in the fall were intermediate to summer and winter, consistent with the transitional nature of this phase. A seasonal analysis revealed that the relative abundances of protein spots were mainly winter-increased. The winter-elevated proteins were involved in fatty acid catabolism and protein folding, whereas the winter-depleted proteins included those that degrade branched-chain amino acids. To identify further state-dependent changes, protein spots were re-evaluated with respect to specific physiological state, confirming the predominance of seasonal differences. Additionally, chaperone and heat shock proteins increased in winter, including HSPA4, HSPB6, and HSP90AB1, which have known roles in protecting against ischemia-reperfusion injury and apoptosis. The most significant and greatest fold change observed was a disappearance of phospho-cofilin 2 at low body temperature, likely a strategy to preserve ATP. The robust summer-to-winter seasonal proteomic shift implies that a winter-protected state is orchestrated before prolonged torpor ensues. Additionally, the general preservation of the proteome during winter hibernation and an increase of stress response proteins, together with dephosphorylation of cofilin 2, highlight the importance of ATP-conserving mechanisms for winter cardioprotection. PMID:21914784

  12. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

    2015-10-30

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  13. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    International Nuclear Information System (INIS)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-01-01

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

  14. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  15. Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

    DEFF Research Database (Denmark)

    Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

    2018-01-01

    identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

  16. Dynamic changes of histone H3 marks during Caenorhabditis elegans lifecycle revealed by middle-down proteomics

    DEFF Research Database (Denmark)

    Sidoli, Simone; Vandamme, Julien; Elisabetta Salcini, Anna

    2016-01-01

    We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval......-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during...... that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms. This article is protected by copyright. All...

  17. Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

    Science.gov (United States)

    Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

    2017-03-01

    In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

    Science.gov (United States)

    Du, Jing; Guo, Shirong; Sun, Jin; Shu, Sheng

    2018-05-01

    The mechanism of exogenous Spd-induced Ca(NO 3 ) 2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO 3 ) 2 stress resistance. Ca(NO 3 ) 2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO 3 ) 2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO 3 ) 2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO 3 ) 2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO 3 ) 2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H 2 O 2 and MDA, were detected after exogenous Spd application during Ca(NO 3 ) 2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO 3 ) 2 stress tolerance mediated by exogenous Spd.

  19. Quantitative Proteomic Analysis Reveals that Antioxidation Mechanisms Contribute to Cold Tolerance in Plantain (Musa paradisiaca L.; ABB Group) Seedlings*

    Science.gov (United States)

    Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A.; Chen, Wei; Yang, Yong; Rose, Jocelyn K. C.; Zhang, Sheng; Yi, Gan-Jun

    2012-01-01

    Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

  20. Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiaca L.; ABB Group) seedlings.

    Science.gov (United States)

    Yang, Qiao-Song; Wu, Jun-Hua; Li, Chun-Yu; Wei, Yue-Rong; Sheng, Ou; Hu, Chun-Hua; Kuang, Rui-Bin; Huang, Yong-Hong; Peng, Xin-Xiang; McCardle, James A; Chen, Wei; Yang, Yong; Rose, Jocelyn K C; Zhang, Sheng; Yi, Gan-Jun

    2012-12-01

    Banana and its close relative, plantain are globally important crops and there is considerable interest in optimizing their cultivation. Plantain has superior cold tolerance compared with banana and a thorough understanding of the molecular mechanisms and responses of plantain to cold stress has great potential value for developing cold tolerant banana cultivars. In this study, we used iTRAQ-based comparative proteomic analysis to investigate the temporal responses of plantain to cold stress. Plantain seedlings were exposed for 0, 6, and 24 h of cold stress at 8 °C and subsequently allowed to recover for 24 h at 28 °C. A total of 3477 plantain proteins were identified, of which 809 showed differential expression from the three treatments. The majority of differentially expressed proteins were predicted to be involved in oxidation-reduction, including oxylipin biosynthesis, whereas others were associated with photosynthesis, photorespiration, and several primary metabolic processes, such as carbohydrate metabolic process and fatty acid beta-oxidation. Western blot analysis and enzyme activity assays were performed on seven differentially expressed, cold-response candidate plantain proteins to validate the proteomics data. Similar analyses of the seven candidate proteins were performed in cold-sensitive banana to examine possible functional conservation, and to compare the results to equivalent responses between the two species. Consistent results were achieved by Western blot and enzyme activity assays, demonstrating that the quantitative proteomics data collected in this study are reliable. Our results suggest that an increase of antioxidant capacity through adapted ROS scavenging capability, reduced production of ROS, and decreased lipid peroxidation contribute to molecular mechanisms for the increased cold tolerance in plantain. To the best of our knowledge, this is the first report of a global investigation on molecular responses of plantain to cold stress by

  1. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

    Science.gov (United States)

    Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman

    2012-02-02

    Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Proteomic analysis of Bifidobacterium longum subsp. infantis reveals the metabolic insight on consumption of prebiotics and host glycans.

    Directory of Open Access Journals (Sweden)

    Jae-Han Kim

    Full Text Available Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO. To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

  3. The Urine Proteome Profile Is Different in Neuromyelitis Optica Compared to Multiple Sclerosis: A Clinical Proteome Study.

    Directory of Open Access Journals (Sweden)

    Helle H Nielsen

    Full Text Available Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO, NMO spectrum disorders (NMO-SD and multiple sclerosis (MS. Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS.The proteins in urine samples from anti-aquaporin 4 (AQP4 seropositive NMO/NMO-SD patients (n = 32, patients with MS (n = 46 and healthy subjects (HS, n = 31 were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig in the urine were validated by nephelometry in an independent cohort (n = 9-10 pr. groups.The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002. Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD.The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS.

  4. Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome

    Directory of Open Access Journals (Sweden)

    Marianna Caterino

    2018-03-01

    Full Text Available Background:/Aims: Renal disease is a common cause of morbidity in patients with Bardet-Biedl syndrome (BBS, however the severity of kidney dysfunction is highly variable. To date, there is little information on the pathogenesis, the risk and predictor factors for poor renal outcome in this setting. The present study aims to analyze the spectrum of urinary proteins in BBS patients, in order to potentially identify 1 disease-specific proteomic profiles that may differentiate the patients from normal subjects; 2 urinary markers of renal dysfunction. Methods: Fourteen individuals (7 males and 7 females with a clinical diagnosis of BBS have been selected in this study. A pool of 10 aged-matched males and 10 aged-matched females have been used as controls for proteomic analysis. The glomerular filtration rate (eGFR has been estimated using the CKD-EPI formula. Variability of eGFR has been retrospectively assessed calculating average annual eGFR decline (ΔeGFR in a mean follow-up period of 4 years (3-7. Results: 42 proteins were significantly over- or under-represented in BBS patients compared with controls; the majority of these proteins are involved in fibrosis, cell adhesion and extracellular matrix organization. Statistic studies revealed a significant correlation between urine fibronectin (u-FN (r2=0.28; p<0.05, CD44 antigen (r2 =0.35; p<0.03 and lysosomal alfa glucosidase ( r20.27; p<0.05 abundance with the eGFR. In addition, u-FN (r2 =0.2389; p<0.05 was significantly correlated with ΔeGFR. Conclusion: The present study demonstrates that urine proteome of BBS patients differs from that of normal subjects; in addition, kidney dysfunction correlated with urine abundance of known markers of renal fibrosis.

  5. Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

    Science.gov (United States)

    Quecine, Maria Carolina; Leite, Thiago Falda; Bini, Andressa Peres; Regiani, Thais; Franceschini, Lívia Maria; Budzinski, Ilara Gabriela Frasson; Marques, Felipe Garbelini; Labate, Mônica Teresa Veneziano; Guidetti-Gonzalez, Simone; Moon, David Henry; Labate, Carlos Alberto

    2016-01-01

    Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

  6. Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants.

    Science.gov (United States)

    Yu, Wencheng; Chen, Zhen; Shen, Liang; Wang, Yuanpeng; Li, Qingbiao; Yan, Shan; Zhong, Chuan-Jian; He, Ning

    2016-04-01

    Some bioflocculants composed of extracellular polymeric substances are produced under peculiar conditions. Bacillus licheniformis CGMCC2876 is a microorganism that secretes both extracellular polysaccharides (EPS) and poly-gamma-glutamic acid (γ-PGA) under stress conditions. In this work, SWATH acquisition LC-MS/MS method was adopted for differential proteomic analysis of B. licheniformis, aiming at determining the bacterial stress mechanism. Compared with LB culture, 190 differentially expressed proteins were identified in B. licheniformis CGMCC2876 cultivated in EPS culture, including 117 up-regulated and 73 down-regulated proteins. In γ-PGA culture, 151 differentially expressed proteins, 89 up-regulated and 62 down-regulated, were found in the cells. Up-regulated proteins involved in amino acid biosynthesis were found to account for 43% and 41% of the proteomes in EPS and γ-PGA cultivated cells, respectively. Additionally, a series of proteins associated with amino acid degradation were found to be repressed under EPS and γ-PGA culture conditions. Transcriptional profiling via the qPCR detection of selected genes verified the proteomic analysis. Analysis of free amino acids in the bacterial cells further suggested the presence of amino acid starvation conditions. EPS or γ-PGA was synthesized to alleviate the effect of amino acid limitation in B. licheniformis. This study identified a stress response mechanism in the synthesis of macromolecules in B. licheniformis, providing potential culture strategies to improve the production of two promising bioflocculants. © 2015 Wiley Periodicals, Inc.

  7. Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

    Directory of Open Access Journals (Sweden)

    Maria Carolina Quecine

    Full Text Available Puccinia psidii sensu lato (s.l. is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava and eucalyptus leaves (PpEucalyptus. NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

  8. Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

    Science.gov (United States)

    El-Bebany, Ahmed F; Rampitsch, Christof; Daayf, Fouad

    2010-01-01

    Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

  9. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Lyon, David; Young, Clifford

    2017-01-01

    that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

  10. Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Chin, Mark H.; Qian, Weijun; Wang, Haixing; Petyuk, Vladislav A.; Bloom, Joshua S.; Sforza, Daniel M.; Lacan, Goran; Liu, Dahai; Khan, Arshad H.; Cantor, Rita M.; Bigelow, Diana J.; Melega, William P.; Camp, David G.; Smith, Richard D.; Smith, Desmond J.

    2008-02-10

    The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.

  11. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    Science.gov (United States)

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  12. Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

    Science.gov (United States)

    Alikhani, Mehdi; Mirzaei, Mehdi; Sabbaghian, Marjan; Parsamatin, Pouria; Karamzadeh, Razieh; Adib, Samane; Sodeifi, Niloofar; Gilani, Mohammad Ali Sadighi; Zabet-Moghaddam, Masoud; Parker, Lindsay; Wu, Yunqi; Gupta, Vivek; Haynes, Paul A; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2017-06-06

    Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. The obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated

  13. Cross-species global proteomics reveals conserved and unique processes in Phytophthora sojae and P. ramorum

    Energy Technology Data Exchange (ETDEWEB)

    Savidor, Alon [ORNL; Donahoo, Ryan S [ORNL; Hurtado-Gonzales, Oscar [University of Tennessee, Knoxville (UTK); Land, Miriam L [ORNL; Shah, Manesh B [ORNL; Lamour, Kurt H [ORNL; McDonald, W Hayes [ORNL

    2008-08-01

    Phytophthora ramorum and Phytophthora sojae are destructive plant pathogens. Phytophthora sojae has a narrow host range whereas P. ramorum has a wide host range. A global proteomic comparison of the vegetative (mycelium) and infective (germinating-cyst) life-stages of P. sojae and P. ramorum was conducted to identify candidate proteins involved in host range, early infection and vegetative growth. Sixty-two candidates for early infection, 26 candidates for vegetative growth, and numerous proteins that may be involved in defining host specificity were identified. In addition, common life stage proteomic trends between the organisms were observed. In mycelia, proteins involved in transport and metabolism of amino acids, carbohydrates and other small molecules were up-regulated. In the germinating cysts, up-regulated proteins associated with lipid transport and metabolism, cytoskeleton and protein synthesis were observed. It appears that the germinating cyst catabolizes lipid reserves through the -oxidation pathway to drive the extensive protein synthesis necessary to produce the germ tube and initiate infection. Once inside the host, the pathogen switches to vegetative growth, where energy is derived from glycolysis and utilized for synthesis of amino acids and other molecules that assist survival in the plant tissue.

  14. Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

    Science.gov (United States)

    Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

    2015-11-06

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

  15. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    Science.gov (United States)

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  16. Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

    Science.gov (United States)

    Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

    2018-04-10

    Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

  17. Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

    Science.gov (United States)

    García-Berrocoso, Teresa; Llombart, Víctor; Colàs-Campàs, Laura; Hainard, Alexandre; Licker, Virginie; Penalba, Anna; Ramiro, Laura; Simats, Alba; Bustamante, Alejandro; Martínez-Saez, Elena; Canals, Francesc; Sanchez, Jean-Charles; Montaner, Joan

    2018-01-01

    Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets. © 2018 by The American Society for

  18. Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen

    2012-11-02

    Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

  19. A proteomics study of barley powdery mildew haustoria.

    Science.gov (United States)

    Godfrey, Dale; Zhang, Ziguo; Saalbach, Gerhard; Thordal-Christensen, Hans

    2009-06-01

    A number of fungal and oomycete plant pathogens of major economic importance feed on their hosts by means of haustoria, which they place inside living plant cells. The underlying mechanisms are poorly understood, partly due to difficulty in preparing haustoria. We have therefore developed a procedure for isolating haustoria from the barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). We subsequently aimed to understand the molecular mechanisms of haustoria through a study of their proteome. Extracted proteins were digested using trypsin, separated by LC, and analysed by MS/MS. Searches of a custom Bgh EST sequence database and the NCBI-NR fungal protein database, using the MS/MS data, identified 204 haustoria proteins. The majority of the proteins appear to have roles in protein metabolic pathways and biological energy production. Surprisingly, pyruvate decarboxylase (PDC), involved in alcoholic fermentation and commonly abundant in fungi and plants, was absent in our Bgh proteome data set. A sequence encoding this enzyme was also absent in our EST sequence database. Significantly, BLAST searches of the recently available Bgh genome sequence data also failed to identify a sequence encoding this enzyme, strongly indicating that Bgh does not have a gene for PDC.

  20. Long-term heat stress induces the inflammatory response in dairy cows revealed by plasma proteome analysis.

    Science.gov (United States)

    Min, Li; Zheng, Nan; Zhao, Shengguo; Cheng, Jianbo; Yang, Yongxin; Zhang, Yangdong; Yang, Hongjian; Wang, Jiaqi

    2016-03-04

    In this work we employed a comparative proteomic approach to evaluate seasonal heat stress and investigate proteomic alterations in plasma of dairy cows. Twelve lactating Holstein dairy cows were used and the treatments were: heat stress (n = 6) in hot summer (at the beginning of the moderate heat stress) and no heat stress (n = 6) in spring natural ambient environment, respectively. Subsequently, heat stress treatment lasted 23 days (at the end of the moderate heat stress) to investigate the alterations of plasma proteins, which might be employed as long-term moderate heat stress response in dairy cows. Changes in plasma proteins were analyzed by two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Analysis of the properties of the identified proteins revealed that the alterations of plasma proteins were related to inflammation in long-term moderate heat stress. Furthermore, the increase in plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) directly demonstrated that long-term moderate heat stress caused an inflammatory response in dairy cows. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals.

    Science.gov (United States)

    Himaya, S W A; Marí, Frank; Lewis, Richard J

    2018-01-10

    Cone snail venoms have separately evolved for predation and defense. Despite remarkable inter- and intra-species variability, defined sets of synergistic venom peptides (cabals) are considered essential for prey capture by cone snails. To better understand the role of predatory cabals in cone snails, we used a high-throughput proteomic data mining and visualisation approach. Using this approach, the relationship between the predatory venom peptides from nine C. purpurascens was systematically analysed. Surprisingly, potentially synergistic levels of κ-PVIIA and δ-PVIA were only identified in five of nine specimens. In contrast, the remaining four specimens lacked significant levels of these known excitotoxins and instead contained high levels of the muscle nAChR blockers ψ-PIIIE and αA-PIVA. Interestingly, one of nine specimens expressed both cabals, suggesting that these sub-groups might represent inter-breeding sub-species of C. purpurascens. High throughput cluster analysis also revealed these two cabals clustered with distinct groups of venom peptides that are presently uncharacterised. This is the first report showing that the cone snails of the same species can deploy two separate and distinct predatory cabals for prey capture and shows that the cabals deployed by this species can be more complex than presently realized. Our semi-automated proteomic analysis facilitates the deconvolution of complex venoms to identify co-evolved families of peptides and help unravel their evolutionary relationships in complex venoms.

  2. Proteomic Analysis Reveals the Deregulation of Inflammation-Related Proteins in Acupuncture-Treated Rats with Asthma Onset

    Directory of Open Access Journals (Sweden)

    Yu-Dong Xu

    2012-01-01

    Full Text Available Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE and mass-spectrometry- (MS- based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11 and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE. These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

  3. Transcriptomic and Proteomic Profiling Revealed High Proportions of Odorant Binding and Antimicrobial Defense Proteins in Olfactory Tissues of the House Mouse

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    Barbora Kuntová

    2018-02-01

    Full Text Available Mammalian olfaction depends on chemosensory neurons of the main olfactory epithelia (MOE, and/or of the accessory olfactory epithelia in the vomeronasal organ (VNO. Thus, we have generated the VNO and MOE transcriptomes and the nasal cavity proteome of the house mouse, Mus musculus musculus. Both transcriptomes had low levels of sexual dimorphisms, while the soluble proteome of the nasal cavity revealed high levels of sexual dimorphism similar to that previously reported in tears and saliva. Due to low levels of sexual dimorphism in the olfactory receptors in MOE and VNO, the sex-specific sensing seems less likely to be dependent on receptor repertoires. However, olfaction may also depend on a continuous removal of background compounds from the sites of detection. Odorant binding proteins (OBPs are thought to be involved in this process and in our study Obp transcripts were most expressed along other lipocalins (e.g., Lcn13, Lcn14 and antimicrobial proteins. At the level of proteome, OBPs were highly abundant with only few being sexually dimorphic. We have, however, detected the major urinary proteins MUP4 and MUP5 in males and females and the male-biased central/group-B MUPs that were thought to be abundant mainly in the urine. The exocrine gland-secreted peptides ESP1 and ESP22 were male-biased but not male-specific in the nose. For the first time, we demonstrate that the expression of nasal lipocalins correlates with antimicrobial proteins thus suggesting that their individual variation may be linked to evolvable mechanisms that regulate natural microbiota and pathogens that regularly enter the body along the ‘eyes-nose-oral cavity’ axis.

  4. Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone.

    Science.gov (United States)

    Meier, Samuel M; Muqaku, Besnik; Ullmann, Ronald; Bileck, Andrea; Kreutz, Dominique; Mader, Johanna C; Knasmüller, Siegfried; Gerner, Christopher

    2015-01-01

    Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac

  5. Proteomic analysis reveals the important roles of alpha-5-collagen and ATP5β during skin ulceration syndrome progression of sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Zhao, Zelong; Jiang, Jingwei; Pan, Yongjia; Sun, Hongjuan; Guan, Xiaoyan; Gao, Shan; Chen, Zhong; Dong, Ying; Zhou, Zunchun

    2018-03-20

    Apostichopus japonicus is one of the most important aquaculture species in China. Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease affected the development of A. japonicus culture industry. To better understand the response mechanisms of A. japonicus during SUS progression, the protein variations in the body wall of A. japonicus at different stages of SUS were investigated by a comparative proteomic approach based on isobaric tags for relative and absolute quantification. A total of 1449 proteins were identified from the samples at different SUS stages. Among these proteins, 145 proteins were differentially expressed in the SUS-related samples compared to those of healthy A. japonicus. These differentially expressed proteins involved a wide range of functions. Among these differentially expressed proteins, only two proteins, alpha-5-collagen and an unknown function protein, were differentially expressed during the whole progression of SUS compared with healthy A. japonicus. In addition, ATP synthase subunit beta (ATP5β) interacted with a variety of proteins with different functions during the SUS progression. These results implied that alpha-5-collagen and ATP5β could play important roles during the SUS progression of A. japonicus. Our study provided a new sight to understand the molecular responses of sea cucumber during the SUS progression and accumulated data for the prevention of SUS in sea cucumber aquaculture. The current study aimed to reveal how the body wall of Apostichopus japonicus response to skin ulceration syndrome (SUS). To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile of sea cucumber during the whole SUS progression. By analyzing the expression differences of the proteome via isobaric labeling-based quantitative proteomic, we identified some proteins which may play important roles during the SUS progression. According to the enrichment analyses of these

  6. Proteomic analysis of Taenia hydatigena cyst fluid reveals unique internal microenvironment.

    Science.gov (United States)

    Zheng, Yadong

    2017-12-01

    Taenia hydatigena is a parasitic flatworm that is widely distributed around the world. Using MS/MS, the proteome of T. hydatigena cyst fluid (CF) was profiled and a total of 520 proteins were identified, 430 of which were of sheep origin. T. hydatigena shared 37 parasite-origin and 109 host-origin CF proteins with Echinococcus granulosus. Compared with E. granulosus, T. hydatigena had much more CF proteins associated with amino acid synthesis and complement cascades. In addition, glutamate metabolism and anti-oxidative reactions were identified as relatively more important events. These results suggest that T. hydatigena metacestodes have internal microenvironment with special immune and oxidative conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Proteomic and Physiological Analyses Reveal Putrescine Responses in Roots of Cucumber Stressed by NaCl

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    Yinghui Yuan

    2016-07-01

    Full Text Available Soil salinity is a major environmental constraint that threatens agricultural productivity. Different strategies have been developed to improve crop salt tolerance, among which the effects of polyamines have been well reported. To gain a better understanding of the cucumber (Cucumis sativus L. responses to NaCl and unravel the underlying mechanism of exogenous putrescine (Put alleviating salt-induced damage, comparative proteomic analysis was conducted on cucumber roots treated with NaCl and/or Put for 7 days. The results showed that exogenous Put restored the root growth inhibited by NaCl. 62 differentially expressed proteins implicated in various biological processes were successfully identified by MALDI-TOF/TOF MS. The four largest categories included proteins involved in defense response (24.2%, protein metabolism (24.2%, carbohydrate metabolism (19.4% and amino acid metabolism (14.5%. Exogenous Put up-regulated most identified proteins involved in carbohydrate metabolism, implying an enhancement in energy generation. Proteins involved in defense response and protein metabolism were differently regulated by Put, which indicated the roles of Put in stress resistance and proteome rearrangement. Put also increased the abundance of proteins involved in amino acid metabolism. Meanwhile, physiological analysis showed that Put could further up-regulated the levels of free amino acids in salt stressed-roots. In addition, Put also improved endogenous polyamines contents by regulating the transcription levels of key enzymes in polyamine metabolism. Taken together, these results suggest that Put may alleviate NaCl-induced growth inhibition through degradation of misfolded/damaged proteins, activation of stress defense, and the promotion of carbohydrate metabolism to generate more energy.

  8. Proteomics of red and white corolla limbs in petunia reveals a novel function of the anthocyanin regulator ANTHOCYANIN1 in determining flower longevity.

    Science.gov (United States)

    Prinsi, Bhakti; Negri, Alfredo S; Quattrocchio, Francesca M; Koes, Ronald E; Espen, Luca

    2016-01-10

    The Petunia hybrida ANTHOCYANIN1 (AN1) gene encodes a transcription factor that regulates both the expression of genes involved in anthocyanin synthesis and the acidification of the vacuolar lumen in corolla epidermal cells. In this work, the comparison between the red flowers of the R27 line with the white flowers of the isogenic an1 mutant line W225 showed that the AN1 gene has further pleiotropic effects on flavonoid biosynthesis as well as on distant physiological traits. The proteomic profiling showed that the an1 mutation was associated to changes in accumulation of several proteins, affecting both anthocyanin synthesis and primary metabolism. The flavonoid composition study confirmed that the an1 mutation provoked a broad attenuation of the entire flavonoid pathway, probably by indirect biochemical events. Moreover, proteomic changes and variation of biochemical parameters revealed that the an1 mutation induced a delay in the onset of flower senescence in W225, as supported by the enhanced longevity of the W225 flowers in planta and the loss of sensitivity of cut flowers to sugar. This study suggests that AN1 is possibly involved in the perception and/or transduction of ethylene signal during flower senescence. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Proteomic analysis reveals age-related changes in tendon matrix composition, with age- and injury-specific matrix fragmentation.

    Science.gov (United States)

    Peffers, Mandy J; Thorpe, Chavaunne T; Collins, John A; Eong, Robin; Wei, Timothy K J; Screen, Hazel R C; Clegg, Peter D

    2014-09-12

    Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

    Science.gov (United States)

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-10-30

    Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Comparative proteomic and metabolomic analysis of Streptomyces tsukubaensis reveals the metabolic mechanism of FK506 overproduction by feeding soybean oil.

    Science.gov (United States)

    Wang, Jun; Liu, Huanhuan; Huang, Di; Jin, Lina; Wang, Cheng; Wen, Jianping

    2017-03-01

    FK506 (tacrolimus) is a 23-membered polyketide macrolide that possesses powerful immunosuppressant activity. In this study, feeding soybean oil into the fermentation culture of Streptomyces tsukubaensis improved FK506 production by 88.8%. To decipher the overproduction mechanism, comparative proteomic and metabolomic analysis was carried out. A total of 72 protein spots with differential expression in the two-dimensional gel electrophoresis (2-DE) were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), and 66 intracellular metabolites were measured by gas chromatography-mass spectrometer (GC-MS). The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation, translation, and stress response, suggesting a better intracellular metabolic environment for the synthesis of FK506. Based on these analyses, 20 key metabolites and precursors of FK506 were supplemented into the soybean oil medium. Among them, lysine, citric acid, shikimic acid, and malonic acid performed excellently for promoting the FK506 production and biomass. Especially, the addition of malonic acid achieved the highest FK506 production, which was 1.56-fold of that in soybean oil medium and 3.05-fold of that in initial medium. This report represented the first comprehensive study on the comparative proteomics and metabolomics applied in S. tsukubaensis, and it would be a rational guidance to further strengthen the FK506 production.

  12. Proteomic analysis of maize grain development using iTRAQ reveals temporal programs of diverse metabolic processes.

    Science.gov (United States)

    Yu, Tao; Li, Geng; Dong, Shuting; Liu, Peng; Zhang, Jiwang; Zhao, Bin

    2016-11-04

    Grain development in maize is an essential process in the plant's life cycle and is vital for use of the plant as a crop for animals and humans. However, little is known regarding the protein regulatory networks that control grain development. Here, isobaric tag for relative and absolute quantification (iTRAQ) technology was used to analyze temporal changes in protein expression during maize grain development. Maize grain proteins and changes in protein expression at eight developmental stages from 3 to 50 d after pollination (DAP) were performed using iTRAQ-based proteomics. Overall, 4751 proteins were identified; 2639 of these were quantified and 1235 showed at least 1.5-fold changes in expression levels at different developmental stages and were identified as differentially expressed proteins (DEPs). The DEPs were involved in different cellular and metabolic processes with a preferential distribution to protein synthesis/destination and metabolism categories. A K-means clustering analysis revealed coordinated protein expression associated with different functional categories/subcategories at different development stages. Our results revealed developing maize grain display different proteomic characteristics at distinct stages, such as numerous DEPs for cell growth/division were highly expressed during early stages, whereas those for starch biosynthesis and defense/stress accumulated in middle and late stages, respectively. We also observed coordinated expression of multiple proteins of the antioxidant system, which are essential for the maintenance of reactive oxygen species (ROS) homeostasis during grain development. Particularly, some DEPs, such as zinc metallothionein class II, pyruvate orthophosphate dikinase (PPDK) and 14-3-3 proteins, undergo major changes in expression at specific developmental stages, suggesting their roles in maize grain development. These results provide a valuable resource for analyzing protein function on a global scale and also

  13. Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta.

    Directory of Open Access Journals (Sweden)

    Kui Shi

    Full Text Available Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs and proteins (DEPs, respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

  14. The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats.

    Science.gov (United States)

    Woo, Patrick C Y; Lau, Susanna K P; Tse, Herman; Teng, Jade L L; Curreem, Shirly O T; Tsang, Alan K L; Fan, Rachel Y Y; Wong, Gilman K M; Huang, Yi; Loman, Nicholas J; Snyder, Lori A S; Cai, James J; Huang, Jian-Dong; Mak, William; Pallen, Mark J; Lok, Si; Yuen, Kwok-Yung

    2009-03-01

    Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish-borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors-such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases-are present. Proteomes of L. hongkongensis HLHK9 cultured at 37 degrees C (human body temperature) and 20 degrees C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)-NAGK-20 and NAGK-37-in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20 degrees C, whereas NAGK-37 showed higher expression at 37 degrees C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other

  15. Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

    Science.gov (United States)

    Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

    2017-04-01

    Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  16. A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

    Science.gov (United States)

    Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

    2017-07-11

    Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT

  17. Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

    Science.gov (United States)

    George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

    2015-09-01

    Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli.

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    Yanlong Wang

    Full Text Available We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli, Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cDNA of Ds-26-16 using the iTRAQ (isobaric tags for relative and absolute quantification approach. A total of 1,610 proteins were identified, which comprised 39.4% of the whole proteome. Of the 559 differential proteins, 259 were up-regulated and 300 were down-regulated. GO (gene ontology and KEGG (Kyoto encyclopedia of genes and genomes enrichment analyses identified 202 major proteins, including those involved in amino acid and organic acid metabolism, energy metabolism, carbon metabolism, ROS (reactive oxygen species scavenging, membrane proteins and ABC (ATP binding cassette transporters, and peptidoglycan synthesis, as well as 5 up-regulated transcription factors. Our iTRAQ data suggest that Ds-26-16 up-regulates the transcription factors in E. coli to enhance salt resistance through osmotic balance, energy metabolism, and oxidative stress protection. Changes in the proteome were also observed in E. coli overexpressing the ORF (open reading frame of Ds-26-16. Furthermore, pH, nitric oxide and glycerol content analyses indicated that Ds-26-16 overexpression increases nitric oxide content but has no effect on glycerol content, thus confirming that enhanced nitric oxide synthesis via lower intercellular pH was one of the mechanisms by which Ds-26-16 confers salt tolerance to E. coli.

  19. Proteomic analysis of tissue from α1,3-galactosyltransferase knockout mice reveals that a wide variety of proteins and protein fragments change expression level.

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    Louise Thorlacius-Ussing

    Full Text Available A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient's blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes.

  20. Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

    Science.gov (United States)

    Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

    2016-11-01

    The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Mechanism of cisplatin proximal tubule toxicity revealed by integrating transcriptomics, proteomics, metabolomics and biokinetics.

    Science.gov (United States)

    Wilmes, Anja; Bielow, Chris; Ranninger, Christina; Bellwon, Patricia; Aschauer, Lydia; Limonciel, Alice; Chassaigne, Hubert; Kristl, Theresa; Aiche, Stephan; Huber, Christian G; Guillou, Claude; Hewitt, Philipp; Leonard, Martin O; Dekant, Wolfgang; Bois, Frederic; Jennings, Paul

    2015-12-25

    Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of solid tumours. The major dose-limiting factor is nephrotoxicity, in particular in the proximal tubule. Here, we use an integrated omics approach, including transcriptomics, proteomics and metabolomics coupled to biokinetics to identify cell stress response pathways induced by cisplatin. The human renal proximal tubular cell line RPTEC/TERT1 was treated with sub-cytotoxic concentrations of cisplatin (0.5 and 2 μM) in a daily repeat dose treating regime for up to 14 days. Biokinetic analysis showed that cisplatin was taken up from the basolateral compartment, transported to the apical compartment, and accumulated in cells over time. This is in line with basolateral uptake of cisplatin via organic cation transporter 2 and bioactivation via gamma-glutamyl transpeptidase located on the apical side of proximal tubular cells. Cisplatin affected several pathways including, p53 signalling, Nrf2 mediated oxidative stress response, mitochondrial processes, mTOR and AMPK signalling. In addition, we identified novel pathways changed by cisplatin, including eIF2 signalling, actin nucleation via the ARP/WASP complex and regulation of cell polarization. In conclusion, using an integrated omic approach together with biokinetics we have identified both novel and established mechanisms of cisplatin toxicity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A novel anti-virulence gene revealed by proteomic analysis in Shigella flexneri 2a

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    Ying Tianyi

    2010-06-01

    Full Text Available Abstract Background Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE analysis to measure changes in the expression profile that are induced by a temperature increase. Results The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgTY225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains. Conclusion Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.

  3. Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes

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    Zhang, Qibin; Fillmore, Thomas L.; Schepmoes, Athena A.; Clauss, Therese RW; Gritsenko, Marina A.; Mueller, Patricia W.; Rewers, Marian; Atkinson, Mark A.; Smith, Richard D.; Metz, Thomas O.

    2013-01-14

    Using global liquid chromatography-mass spectrometry (LC-MS)-based proteomics analyses, we identified 24 serum proteins significantly variant between those with type 1 diabetes and healthy controls. Functionally, these proteins represent innate immune responses, the activation cascade of complement, inflammatory responses and blood coagulation. Targeted verification analyses were performed on 52 surrogate peptides representing these proteins with serum samples from an antibody standardization program cohort of 100 healthy control and 50 type 1 diabetic subjects, and 16 peptides were verified having very good discriminating power, with areas under the receiver operator characteristic curve ≥ 0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetic) demonstrated that peptides from platelet basic protein and C1 inhibitor achieved both 100% sensitivity and 100% specificity for classification of samples. The disease specificity of these proteins was assessed using serum from 50 age matched type 2 diabetic individuals, and a subset of proteins, particularly C1 inhibitor were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with type 1 diabetes from healthy control and type 2 diabetes suggests dysregulated innate immune responses may be associated with the development of this disorder.

  4. Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

    Science.gov (United States)

    Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

    2014-11-01

    Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  5. Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

    Science.gov (United States)

    Soman, Kizhake V; Stafford, Susan J; Pazdrak, Konrad; Wu, Zheng; Luo, Xuemei; White, Wendy I; Wiktorowicz, John E; Calhoun, William J; Kurosky, Alexander

    2017-08-04

    Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

  6. Radiation induced changes in proteome of mice jejunum: an in vivo 2DE study

    International Nuclear Information System (INIS)

    Bajaj, Sania; Dutta, Ajaswrata; Gupta, Manju L.

    2014-01-01

    Radiation exposure results in severe damage to biological system, by affecting cellular macromolecules of an individual. Damage to bio-molecules may lead to up/down-regulation of proteins, leading to dysfunction of organs. Gastrointestinal tract a key organ for digestion, absorption and barrier to the luminal bacteria and toxins, is one of the highly sensitive radiosensitive organ. Current study is focused on differential proteomic approach to understand the effect of radiation on intestinal (jejunum) proteins in a time dependent manner. Experiments were carried out initially to determine the appropriate conditions for separation of proteins in GI tissue of non irradiated control male C57BL6/J mice. 8-10 weeks old animals were exposed to 9 Gy (lethal) dose of gamma radiation. Differential expression of gastrointestinal tissue (jejunum) proteome was studied by 2DE at different time intervals. The intensity of protein spots of different treatment groups and control was measured by PD Quest software and the differential expression of respective proteins was calculated manually. Comparison of 2-DE gel images of irradiated jejunum tissue samples showed differential expression of various proteins when compared with untreated samples. A significant upregulation of total protein spots was observed within 1 hr group of 9 Gy radiation exposed sample and maximum down-regulation was evident at 72 hr. Out of 24 spots identified in the irradiated samples, 15 spots were down-regulated, and 3 spots were found missing in 72 hr group of irradiated samples respectively. Time dependent regulation of protein expression in irradiated jejunum was thus prominently evident. The data obtained from the present study has revealed differential radio sensitivity of some of the proteins which certainly have a definite role in inducing major cellular changes after radiation exposure. The finding also suggests that proteomic approach could be a potential tool to access the role of specific

  7. Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics

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    Sijia Wei

    2017-05-01

    Full Text Available Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway, nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein–protein interaction (PPI networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain.

  8. Comparative Transcriptomic and Proteomic Analyses Reveal a FluG-Mediated Signaling Pathway Relating to Asexual Sporulation of Antrodia camphorata.

    Science.gov (United States)

    Li, Hua-Xiang; Lu, Zhen-Ming; Zhu, Qing; Gong, Jin-Song; Geng, Yan; Shi, Jin-Song; Xu, Zheng-Hong; Ma, Yan-He

    2017-09-01

    Medicinal mushroom Antrodia camphorata sporulate large numbers of arthroconidia in submerged fermentation, which is rarely reported in basidiomycetous fungi. Nevertheless, the molecular mechanisms underlying this asexual sporulation (conidiation) remain unclear. Here, we used comparative transcriptomic and proteomic approaches to elucidate possible signaling pathway relating to the asexual sporulation of A. camphorata. First, 104 differentially expressed proteins and 2586 differential cDNA sequences during the culture process of A. camphorata were identified by 2DE and RNA-seq, respectively. By applying bioinformatics analysis, a total of 67 genes which might play roles in the sporulation were obtained, and 18 of these genes, including fluG, sfgA, SfaD, flbA, flbB, flbC, flbD, nsdD, brlA, abaA, wetA, ganB, fadA, PkaA, veA, velB, vosA, and stuA might be involved in a potential FluG-mediated signaling pathway. Furthermore, the mRNA expression levels of the 18 genes in the proposed FluG-mediated signaling pathway were analyzed by quantitative real-time PCR. In summary, our study helps elucidate the molecular mechanisms underlying the asexual sporulation of A. camphorata, and provides also useful transcripts and proteome for further bioinformatics study of this valuable medicinal mushroom. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings.

    Science.gov (United States)

    Sang, Ting; Shan, Xi; Li, Bin; Shu, Sheng; Sun, Jin; Guo, Shirong

    2016-08-01

    Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings. Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

  10. Temporal proteomic analysis reveals defects in small-intestinal development of porcine fetuses with intrauterine growth restriction.

    Science.gov (United States)

    Wang, Xiaoqiu; Lin, Gang; Liu, Chuang; Feng, Cuiping; Zhou, Huaijun; Wang, Taiji; Li, Defa; Wu, Guoyao; Wang, Junjun

    2014-07-01

    The fetus/neonate with intrauterine growth restriction (IUGR) has a high perinatal mortality and morbidity rate, as well as reduced efficiency for nutrients utilization. Our previous studies showed alterations of intestinal proteome in IUGR piglets both at birth and during the nursing period. Considering the potential long-term impacts of fetal programming and substantial increases in amounts of amniotic fluid nutrients from mid-gestation in pigs, the present study involved IUGR porcine fetuses from days 60 to 110 of gestation (mid to late gestation). We identified 59 differentially expressed proteins in the fetal small intestine that are related to intestinal growth, development and reprogramming. Our results further indicated increased abundances of proteins and enzymes associated with oxidative stress, apoptosis and protein degradation, as well as decreased abundances of proteins that are required for maintenance of cell structure and motility, absorption and transport of nutrients, energy metabolism, and protein synthesis in the fetal gut. Moreover, IUGR from middle to late gestation was associated with reduced expression of intestinal proteins that participate in regulation of gene expression and signal transduction. Collectively, these findings provide the first evidence for altered proteomes in the small intestine of IUGR fetuses, thereby predisposing the gut to metabolic defects during gestation and neonatal periods. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

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    Marlena M Wilson

    Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

  12. Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

    Science.gov (United States)

    Klaeger, Susan; Gohlke, Bjoern; Perrin, Jessica; Gupta, Vipul; Heinzlmeir, Stephanie; Helm, Dominic; Qiao, Huichao; Bergamini, Giovanna; Handa, Hiroshi; Savitski, Mikhail M; Bantscheff, Marcus; Médard, Guillaume; Preissner, Robert; Kuster, Bernhard

    2016-05-20

    Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

  13. Proteomic responses reveal the differential effects induced by cadmium in mussels Mytilus galloprovincialis at early life stages.

    Science.gov (United States)

    Xu, Lanlan; Peng, Xiao; Yu, Deliang; Ji, Chenglong; Zhao, Jianmin; Wu, Huifeng

    2016-08-01

    Cadmium (Cd) has become an important metal contaminant and posed severe risk on the organisms in the coastal environments of the Bohai Sea. Marine mussel Mytilus galloprovincialis is widely distributed along the Bohai coast and consumed as seafood by local residents. Evidences indicate that the early stages of marine organisms are more sensitive to metal contaminants. In this study, we applied two-dimensional electrophoresis-based proteomics to characterize the biological effects of Cd (50 μg L(-1)) in the early life stages (D-shape larval and juvenile) of mussels. The different proteomic responses demonstrated the differential responsive mechanisms to Cd exposure in these two early life stages of mussels. In details, results indicated that Cd mainly induced immune and oxidative stresses in both D-shape larval and juvenile mussels via different pathways. In addition, the significant up-regulation of triosephosphate isomerase and metallothionein confirmed the enhanced energy demand and mobilized detoxification mechanism in D-shape larval mussels exposed to Cd. In juvenile mussels, Cd exposure also induced clear apoptosis. Overall, this work suggests that Cd is a potential immune toxicant to mussel M. galloprovincialis at early life stages. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

    Science.gov (United States)

    Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

    2016-12-01

    Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. PROTEOMIC PROFILE REVEALS THE DIVERSITY AND COMPLEXITY OF LEAF PROTEINS IN SPINACH (BETA VULGARIS VAR. ALL GREEN

    Directory of Open Access Journals (Sweden)

    Sudip Ghosh

    2016-06-01

    Full Text Available Leaf is a source organ that serves dual function in photosynthesis and transpiration. As a primary interface between plant and ecosystem, it performs a range of biological processes from carbon assimilation and metabolite partitioning to plant productivity. Basic features of the leaf functionality are conserved in angiosperms exhibiting common and unique characteristics. Spinach has been the model crop for studying leaf function, primarily photosynthesis. It is a reservoir of several hundreds of primary and secondary biomolecules. To better understand the molecular basis for photochemical reaction and metabolic partitioning, we developed leaf proteome of Indian spinach (Beta vulgaris var. all green. LC-ESI-MS/MS analysis identified 639 proteins exhibiting discrete molecular features and functions, including photosynthesis, transpiration, gaseous exchange, transport, redox status, cell defense, and floral induction besides the presence of proteins with unknown function. This represents the first comprehensive foliage proteome of green leafy vegetable. Together, this work provides important insights into the molecular networks underlying spinach leaf biological processes.

  16. Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani.

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    Bhavna Chawla

    Full Text Available Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.

  17. Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

    Science.gov (United States)

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

    2013-07-11

    FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion

  18. Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

    Science.gov (United States)

    Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan

    2015-08-03

    Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis

    Directory of Open Access Journals (Sweden)

    Bhavna Daswani

    2015-01-01

    Full Text Available Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27 distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n=80 using intracellular ELISA confirmed that total HSP27 (tHSP27 as well as phosphorylated HSP27 (pHSP27 was elevated in low BMD condition in both categories (P<0.05. Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P<0.0001 and this was additive in combination with RANKL (receptor activator of NFkB ligand placed in the lower chamber (P=0.05. Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

  20. Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

    Directory of Open Access Journals (Sweden)

    Uta Gey

    Full Text Available The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p. The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p, Lys114 (Kgd2p and Lys102 (Gcv3p, respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

  1. A proteomic style approach to characterize a grass mix product reveals potential immunotherapeutic benefit.

    Science.gov (United States)

    Bullimore, Alan; Swan, Nicola; Alawode, Wemimo; Skinner, Murray

    2011-09-01

    Grass allergy immunotherapies often consist of a mix of different grass extracts, each containing several proteins of different physiochemical properties; however, the subtle contributions of each protein are difficult to elucidate. This study aimed to identify and characterize the group 1 and 5 allergens in a 13 grass extract and to standardize the extraction method. The grass pollens were extracted in isolation and pooled and also in combination and analyzed using a variety of techniques including enzyme-linked immunosorbent assay, liquid chromatog-raphy-mass spectrometry, and sodium dodecyl sulfate-polyacrylam-ide gel electrophoresis. Gold-staining and IgE immunoblotting revealed a high degree of homology of protein bands between the 13 species and the presence of a densely stained doublet at 25-35 kD along with protein bands at approximately 12.5, 17, and 50 kD. The doublet from each grass species demonstrated a high level of group 1 and 5 interspecies homology. However, there were a number of bands unique to specific grasses consistent with evolutionary change and indicative that a grass mix immunotherapeutic could be considered broad spectrum. Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis and IgE immunoblotting showed all 13 grasses share a high degree of homology, particularly in terms of group 1 and 5 allergens. IgE and IgG enzyme-linked immunosorbent assay potencies were shown to be independent of extraction method.

  2. Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis.

    Science.gov (United States)

    Dahlhoff, Maik; Fröhlich, Thomas; Arnold, Georg J; Müller, Udo; Leonhardt, Heinrich; Zouboulis, Christos C; Schneider, Marlon R

    2015-03-01

    Lipid metabolism depends on lipid droplets (LD), cytoplasmic structures surrounded by a protein-rich phospholipid monolayer. Although lipid synthesis is the hallmark of sebaceous gland cell differentiation, the LD-associated proteins of sebocytes have not been evaluated systematically. The LD fraction of SZ95 sebocytes was collected by density gradient centrifugation and associated proteins were analyzed by nanoliquid chromatography/tandem mass spectrometry. 54 proteins were significantly enriched in LD fractions, and 6 of them have not been detected previously in LDs. LD fractions contained high levels of typical LD-associated proteins as PLIN2/PLIN3, and most proteins belonged to functional categories characteristic for LD-associated proteins, indicating a reliable dataset. After confirming expression of transcripts encoding the six previously unidentified proteins by qRT-PCR in SZ95 sebocytes and in another sebocyte line (SebE6E7), we focused on two of these proteins, ALDH1A3 and EPHX4. While EPHX4 was localized almost exclusively on the surface of LDs, ALDH1A3 showed a more widespread localization that included additional cytoplasmic structures. siRNA-mediated downregulation revealed that depletion of EPHX4 increases LD size and sebaceous lipogenesis. Further studies on the roles of these proteins in sebocyte physiology and sebaceous lipogenesis may indicate novel strategies for the therapy of sebaceous gland-associated diseases such as acne. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-06

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.

  4. Studying Different Clinical Syndromes Of Paediatric Severe Malaria Using Plasma Proteomics

    KAUST Repository

    Ramaprasad, Abhinay

    2012-01-01

    challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory

  5. Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Kasinath Viswanathan

    Full Text Available Human cytomegalovirus (HCMV depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC, membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.

  6. ABRF-PRG07: advanced quantitative proteomics study.

    Science.gov (United States)

    Falick, Arnold M; Lane, William S; Lilley, Kathryn S; MacCoss, Michael J; Phinney, Brett S; Sherman, Nicholas E; Weintraub, Susan T; Witkowska, H Ewa; Yates, Nathan A

    2011-04-01

    A major challenge for core facilities is determining quantitative protein differences across complex biological samples. Although there are numerous techniques in the literature for relative and absolute protein quantification, the majority is nonroutine and can be challenging to carry out effectively. There are few studies comparing these technologies in terms of their reproducibility, accuracy, and precision, and no studies to date deal with performance across multiple laboratories with varied levels of expertise. Here, we describe an Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) study based on samples composed of a complex protein mixture into which 12 known proteins were added at varying but defined ratios. All of the proteins were present at the same concentration in each of three tubes that were provided. The primary goal of this study was to allow each laboratory to evaluate its capabilities and approaches with regard to: detection and identification of proteins spiked into samples that also contain complex mixtures of background proteins and determination of relative quantities of the spiked proteins. The results returned by 43 participants were compiled by the PRG, which also collected information about the strategies used to assess overall performance and as an aid to development of optimized protocols for the methodologies used. The most accurate results were generally reported by the most experienced laboratories. Among laboratories that used the same technique, values that were closer to the expected ratio were obtained by more experienced groups.

  7. Quantitative proteome-level analysis of paulownia witches’ broom disease with methyl methane sulfonate assistance reveals diverse metabolic changes during the infection and recovery processes

    Directory of Open Access Journals (Sweden)

    Zhe Wang

    2017-07-01

    Full Text Available Paulownia witches’ broom (PaWB disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ to quantify and analyze the proteome-level changes among healthy P. fortunei (PF, PaWB-infected P. fortunei (PFI, and PaWB-infected P. fortunei treated with 20 mg L−1 or 60 mg L−1 methyl methane sulfonate (MMS (PFI-20 and PFI-60, respectively. A total of 2,358 proteins were identified. We investigated the proteins profiles in PF vs. PFI (infected process and PFI-20 vs. PFI-60 (recovered process, and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1 carbohydrate and energy metabolism, (2 protein synthesis and degradation, and (3 stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

  8. iTRAQ-Based Proteomic Analysis Reveals Potential Regulation Networks of IBA-Induced Adventitious Root Formation in Apple

    Directory of Open Access Journals (Sweden)

    Chao Lei

    2018-02-01

    Full Text Available Adventitious root (AR formation, which is controlled by endogenous and environmental factors, is indispensable for vegetative asexual propagation. However, comprehensive proteomic data on AR formation are still lacking. The aim of this work was to study indole-3-butyric acid (IBA-induced AR formation in the dwarf apple rootstock ‘T337’. In this study, the effect of IBA on AR formation was analysed. Subsequent to treatment with IBA, both the rooting rate and root length of ‘T337’ increased significantly. An assessment of hormone levels in basal stem cuttings suggested that auxin, abscisic acid, and brassinolide were higher in basal stem cuttings that received the exogenous IBA application; while zeatin riboside, gibberellins, and jasmonic acid were lower than non-treated basal stem cuttings. To explore the underlying molecular mechanism, an isobaric tags for relative and absolute quantification (iTRAQ-based proteomic technique was employed to identify the expression profiles of proteins at a key period of adventitious root induction (three days after IBA treatment. In total, 3355 differentially expressed proteins (DEPs were identified. Many DEPs were closely related to carbohydrate metabolism and energy production, protein homeostasis, reactive oxygen and nitric oxide signaling, and cell wall remodeling biological processes; as well as the phytohormone signaling, which was the most critical process in response to IBA treatment. Further, RT-qPCR analysis was used to evaluate the expression level of nine genes that are involved in phytohormone signaling and their transcriptional levels were mostly in accordance with the protein patterns. Finally, a putative work model was proposed. Our study establishes a foundation for further research and sheds light on IBA-mediated AR formation in apple as well as other fruit rootstock cuttings.

  9. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate

    2012-01-01

    ,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals...

  10. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara

    2013-01-01

    PRDM proteins belong to the SET- domain protein family involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we...... find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......, despite Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, Cohesin and TFIIIC and co...

  11. Physiological and comparative proteomic analysis reveals different drought responses in roots and leaves of drought-tolerant wild wheat (Triticum boeoticum.

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    Hui Liu

    Full Text Available To determine the proteomic-level responses of drought tolerant wild wheat (Triticum boeoticum, physiological and comparative proteomic analyses were conducted using the roots and the leaves of control and short term drought-stressed plants. Drought stress was imposed by transferring hydroponically grown seedlings at the 3-leaf stage into 1/2 Hoagland solution containing 20% PEG-6000 for 48 h. Root and leaf samples were separately collected at 0 (control, 24, and 48 h of drought treatment for analysis. Physiological analysis indicated that abscisic acid (ABA level was greatly increased in the drought-treated plants, but the increase was greater and more rapid in the leaves than in the roots. The net photosynthetic rate of the wild wheat leaves was significantly decreased under short-term drought stress. The deleterious effects of drought on the studied traits mainly targeted photosynthesis. Comparative proteomic analysis identified 98 and 85 differently changed protein spots (DEPs (corresponding to 87 and 80 unique proteins, respectively in the leaves and the roots, respectively, with only 6 mutual unique proteins in the both organs. An impressive 86% of the DEPs were implicated in detoxification and defense, carbon metabolism, amino acid and nitrogen metabolism, proteins metabolism, chaperones, transcription and translation, photosynthesis, nucleotide metabolism, and signal transduction. Further analysis revealed some mutual and tissue-specific responses to short-term drought in the leaves and the roots. The differences of drought-response between the roots and the leaves mainly included that signal sensing and transduction-associated proteins were greatly up-regulated in the roots. Photosynthesis and carbon fixation ability were decreased in the leaves. Glycolysis was down-regulated but PPP pathway enhanced in the roots, resulting in occurrence of complex changes in energy metabolism and establishment of a new homeostasis. Protein metabolism

  12. Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression.

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    Danilo Marimpietri

    Full Text Available Neuroblastoma (NB is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133, basigin (CD147 and B7-H3 (CD276. Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

  13. Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host-parasite interactions.

    Science.gov (United States)

    Cui, Shu-Jian; Xu, Lei-Lei; Zhang, Ting; Xu, Ming; Yao, Jun; Fang, Cai-Yun; Feng, Zheng; Yang, Peng-Yuan; Hu, Wei; Liu, Feng

    2013-06-12

    Cystic hydatid disease is an important zoonosis caused by Echinococcus granulosus infection. The expression profiles of its parasitic life stages and host-Echinococcus interactions remain to be elucidated. Here, we identified 157 adult and 1588 protoscolex proteins (1610 in all), including 1290 novel identifications. Paramyosins and an antigen B (AgB) were the dominant adult proteins. Dog proteins (30) identified in adults indicated diminished local inflammation caused by adult infection. The protoscolex expresses proteins that have been reported to be antigens in other parasites, such as 6-phosphofructokinase and calcineurin B. Pathway analyses suggested that E. granulosus uses both aerobic and anaerobic carbohydrate metabolisms to generate ATP. E. granulosus expresses proteins involved in synthesis and metabolism of lipids or steroids. At least 339 of 390 sheep proteins identified in protoscolex were novel identifications not seen in previous analyses. IgGs and lambda light chains were the most abundant antibody species. Sheep proteins were enriched for detoxification pathways, implying that host detoxification effects play a central role during host-parasite interactions. Our study provides valuable data on E. granulosus expression characteristics, allowing novel insights into the molecular mechanisms involved in host-parasite interactions. In this study, the Echinococcus granulosus adult worm proteome was analyzed for the first time. The protein identification of E. granulosus protoscoleces was extended dramatically. We also identified the most abundant host proteins co-purified with Echinococcus. The results provide useful information pertaining to the molecular mechanisms behind host-Echinococcus interaction and Echinococcus biology. This data also increases the potential for identifying vaccine candidates and new therapeutic targets, and may aid in the development of protein probes for selective and sensitive diagnosis of echinococcosis infection. In

  14. Comparative Proteomics Reveals Differences in Host-Pathogen Interaction between Infectious and Commensal Relationship with Campylobacter jejuni

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    Juan J. Garrido

    2017-04-01

    Full Text Available Campylobacter jejuni is the leading food-borne poisoning in industrialized countries. While the bacteria causes disease in humans, it merely colonizes the gut in poultry or pigs, where seems to establish a commensal relationship. Until now, few studies have been conducted to elucidate the relationship between C. jejuni and its different hosts. In this work, a comparative proteomics approach was used to identify the underlying mechanisms involved in the divergent outcome following C. jejuni infection in human and porcine host. Human (INT-407 and porcine (IPEC-1 intestinal cell lines were infected by C. jejuni for 3 h (T3h and 24 h (T24h. C. jejuni infection prompted an intense inflammatory response at T3h in human intestinal cells, mainly characterized by expression of proteins involved in cell spreading, cell migration and promotion of reactive oxygen species (ROS. Proteomic analysis evidenced significantly regulated biofunctions in human cells related with engulfment and endocytosis, and supported by canonical pathways associated to infection such as caveolar- and clathrin-mediated endocytosis signaling. In porcine IPEC-1 cells, inflammatory response as well as signaling pathways that control cellular functions such as cell migration, endocytosis and cell cycle progression resulted downregulated. These differences in the host response to infection were supported by the different pattern of adhesion and invasion proteins expressed by C. jejuni in human and porcine cells. No marked differences in expression of virulence factors involved in adaptive response and iron acquisition functions were observed. Therefore, the results of this study suggest that both host and pathogen factors are responsible for commensal or infectious character of C. jejuni in different hosts.

  15. Proteomic profiling reveals α1-antitrypsin, α1-microglobulin, and clusterin as preeclampsia-related serum proteins in pregnant women.

    Science.gov (United States)

    Hsu, Te-Yao; Hsieh, T'sang-T'ang; Yang, Kuender D; Tsai, Ching-Chang; Ou, Chia-Yu; Cheng, Bi-Hua; Wong, Yi-Hsun; Hung, Hsuan-Ning; Chou, An-Kuo; Hsiao, Chang-Chun; Lin, Hao

    2015-10-01

    Preeclampsia is a major cause of mortality in pregnant women but the underlying mechanism remains unclear to date. In this study, we attempted to identify candidate proteins that might be associated with preeclampsia in pregnant women by means of proteomics tools. Differentially expressed proteins in serum samples obtained from pregnant women with severe preeclampsia (n = 8) and control participants (n = 8) were identified using two-dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Additional serum samples from 50 normal and 41 pregnant women with severe preeclampsia were analyzed by immunoassay for validation. Ten protein spots were found to be upregulated significantly in women with severe preeclampsia. These protein spots had the peptide mass fingerprints matched to α1-antitrypsin, α1-microglobulin, clusterin, and haptoglobin. Immunoassays in an independent series of serum samples showed that serum α1-antitrypsin, α1-microglobulin, and clusterin levels of severe preeclampsia patients (n = 41) were significantly higher than those in the normal participants (n = 50; α1-antitrypsin 295.95 ± 50.94 mg/dL vs. 259.31 ± 33.90 mg/dL, p = 0.02; α1-microglobulin 0.029 ± 0.004 mg/mL vs. 0.020 ± 0.004 mg/mL, p proteins by proteomics analysis enables further understanding of the pathophysiology of preeclampsia. Further studies are warranted to investigate the role of these biomarkers in prediction of this disease. Copyright © 2015. Published by Elsevier B.V.

  16. Analysis of the variability of human normal urine by 2D-GE reveals a "public" and a "private" proteome.

    Science.gov (United States)

    Molina, Laurence; Salvetat, Nicolas; Ameur, Randa Ben; Peres, Sabine; Sommerer, Nicolas; Jarraya, Fayçal; Ayadi, Hammadi; Molina, Franck; Granier, Claude

    2011-12-10

    The characterization of the normal urinary proteome is steadily progressing and represents a major interest in the assessment of clinical urinary biomarkers. To estimate quantitatively the variability of the normal urinary proteome, urines of 20 healthy people were collected. We first evaluated the impact of the sample conservation temperature on urine proteome integrity. Keeping the urine sample at RT or at +4°C until storage at -80°C seems the best way for long-term storage of samples for 2D-GE analysis. The quantitative variability of the normal urinary proteome was estimated on the 20 urines mapped by 2D-GE. The occurrence of the 910 identified spots was analysed throughout the gels and represented in a virtual 2D gel. Sixteen percent of the spots were found to occur in all samples and 23% occurred in at least 90% of urines. About 13% of the protein spots were present only in 10% or less of the samples, thus representing the most variable part of the normal urinary proteome. Twenty proteins corresponding to a fraction of the fully conserved spots were identified by mass spectrometry. In conclusion, a "public" urinary proteome, common to healthy individuals, seems to coexist with a "private" urinary proteome, which is more specific to each individual. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

  18. Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

    Science.gov (United States)

    Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

    2013-05-10

    Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level

  19. Predicted sub-populations in a marine shrimp proteome as revealed by combined EST and cDNA data from multiple Penaeus species

    Directory of Open Access Journals (Sweden)

    Kotewong Rattanawadee

    2010-11-01

    Full Text Available Abstract Background Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus vannamei, Penaeus monodon, Penaeus (Fenneropenaeus chinensis, and Penaeus (Marsupenaeus japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda, the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used. Findings Similarity searches from EST collections of the 4 shrimp species matched 64% of the protein sequences of the fruit fly, but only 45% of nematode proteins, indicating that the shrimp proteome content is more similar to that of an insect than a nematode. Combined results with 4 additional non-shrimp crustaceans increased matching to 78% of fruit fly and 56% of nematode proteins, suggesting that present shrimp EST collections still lack sequences for many conserved crustacean proteins. Analysis of matching data revealed the presence of 4 EST groups from shrimp, namely sequences for proteins that are both fruit fly-like and nematode-like, fruit fly-like only, nematode-like only, and non-matching. Gene ontology profiles of proteins for the 3 matching EST groups were analyzed. For non-matching ESTs, a small fraction matched protein sequences from other species in the UniProt database, including other crustacean-specific proteins. Conclusions Shrimp ESTs indicated that the shrimp proteome is comprised of sub-populations of proteins similar to those common to both insect and nematode models, those present specifically in either model, or neither. Combining small EST collections from related species to compensate for their

  20. Proteomic profiles reveal age-related changes in coelomic fluid of sea urchin species with different life spans.

    Science.gov (United States)

    Bodnar, Andrea

    2013-05-01

    Sea urchins have a different life history from humans and traditional model organisms used to study the process of aging. Sea urchins grow indeterminately, reproduce throughout their life span and some species have been shown to exhibit negligible senescence with no increase in mortality rate at advanced ages. Despite these properties, different species of sea urchins are reported to have very different natural life spans providing a unique model to investigate cellular mechanisms underlying life span determination and negligible senescence. To gain insight into the biological changes that accompany aging in these animals, proteomic profiles were examined in coelomic fluid from young and old sea urchins of three species with different life spans: short-lived Lytechinus variegatus, long-lived Strongylocentrotus franciscanus and Strongylocentrotus purpuratus which has an intermediate life span. The proteomic profiles of cell-free coelomic fluid were complex with many proteins exhibiting different forms and extensive post-translational modifications. Approximately 20% of the protein spots on 2-D gels showed more than two-fold change with age in each of the species. Changes that are consistent with age in all three species may prove to be useful biomarkers for age-determination for these commercially fished marine invertebrates and also may provide clues to mechanisms of negligible senescence. Among the proteins that change with age, the ectodomain of low-density lipoprotein receptor-related protein 4 (LRP4) was significantly increased in the coelomic fluid of all three sea urchin species suggesting that the Wnt signaling pathway should be further investigated for its role in negligible senescence. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Proteomic Studies of Cholangiocarcinoma and Hepatocellular Carcinoma Cell Secretomes

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    Chantragan Srisomsap

    2010-01-01

    Full Text Available Cholangiocarcinoma (CCA and hepatocellular carcinoma (HCC occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1 and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2, laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

  2. A comparative proteomic study on the effects of metal pollution in oysters Crassostrea hongkongensis.

    Science.gov (United States)

    Xu, Lanlan; Ji, Chenglong; Wu, Huifeng; Tan, Qiaoguo; Wang, Wen-Xiong

    2016-11-15

    The metal pollution has posed great risk on the coastal organisms along the Jiulongjiang Estuary in South China. In this work, two-dimensional electrophoresis-based proteomics was applied to the oysters Crassostrea hongkongensis from metal pollution sites to characterize the proteomic responses to metal pollution. Metal accumulation and proteomic responses indicated that the oysters from BJ site were more severely contaminated than those from FG site. Compared with those oyster samples from the clean site (JZ), metal pollution induced cellular injuries, oxidative and immune stresses in oyster heapatopancreas from both BJ and FG sites via differential metabolic pathways. In addition, metal pollution in BJ site induced disturbance in energy and lipid metabolisms in oysters. Results indicated that cathepsin L and ferritin GF1 might be the biomarkers of As and Fe in oyster C. hongkongensis, respectively. This study demonstrates that proteomics is a useful tool for investigating biological effects induced by metal pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status.

    Science.gov (United States)

    Zachut, Maya

    2015-07-02

    Adipose tissue is a central regulator of metabolism in dairy cows; however, little is known about the association between various proteins in adipose tissue and the metabolic status of peripartum cows. Therefore, the objectives were to (1) examine total protein expression in adipose tissue of dairy cows and (2) identify biomarkers in adipose that are linked to insulin resistance and to cows' metabolic status. Adipose tissue biopsies were obtained from eight multiparous cows at -17 and +4 days relative to parturition. Proteins were analyzed by intensity-based, label-free, quantitative shotgun proteomics (nanoLC-MS/MS). Cows were divided into groups with insulin-resistant (IR) and insulin-sensitive (IS) adipose according to protein kinase B phosphorylation following insulin stimulation. Cows with IR adipose lost more body weight postpartum compared with IS cows. Differential expression of 143 out of 586 proteins was detected in prepartum versus postpartum adipose. Comparing IR to IS adipose revealed differential expression of 18.9% of the proteins; those related to lipolysis (hormone-sensitive lipase, perilipin, monoglycerol lipase) were increased in IR adipose. In conclusion, we found novel biomarkers related to IR in adipose and to metabolic status that could be used to characterize high-yielding dairy cows that are better adapted to peripartum metabolic stress.

  4. Proteomic profiling of human keratinocytes undergoing UVB-induced alternative differentiation reveals TRIpartite Motif Protein 29 as a survival factor.

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    Véronique Bertrand-Vallery

    Full Text Available BACKGROUND: Repeated exposures to UVB of human keratinocytes lacking functional p16(INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. METHODOLOGY/PRINCIPAL FINDINGS: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29. Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCdelta. CONCLUSIONS/SIGNIFICANCE: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.

  5. Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components1[W

    Science.gov (United States)

    Barsan, Cristina; Zouine, Mohamed; Maza, Elie; Bian, Wanping; Egea, Isabel; Rossignol, Michel; Bouyssie, David; Pichereaux, Carole; Purgatto, Eduardo; Bouzayen, Mondher; Latché, Alain; Pech, Jean-Claude

    2012-01-01

    A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome. PMID:22908117

  6. Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

    Science.gov (United States)

    Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Fleming, Joy; Gao, Ding; Bi, Lijun; Ge, Feng

    2015-01-01

    Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs. PMID:25762744

  7. A proteomics study of hyperhomocysteinemia injury of the hippocampal neurons using iTRAQ.

    Science.gov (United States)

    Fang, Min; Wang, Jing; Yan, Han; Zhao, Yan-Xin; Liu, Xue-Yuan

    2014-11-01

    High levels of homocysteine, caused by abnormal methionine metabolism, can induce degeneration of mouse hippocampal neurons. iTRAQ™ technology has been widely used in the field of proteomics research and through employing this technology, the present study identified that hyperhomocysteinemia induced the downregulation of 52 proteins and upregulation of 44 proteins in the mouse hippocampus. Through gene ontology and pathway analysis, the upregulation of components of the cytoskeleton, actin, regulators of focal adhesion, calcium signaling pathways, tight junctions, ErbB and gonadotrophin‑releasing hormone signaling, leukocyte, transendothelial migration, propanoate and pyruvate metabolism, valine, leucine and isoleucine biosynthesis, synthesis and degradation of ketone bodies and benzoate degradation via CoA ligation pathway, was identified. It was additionally verified that tau protein was highly expressed in the hyperhomocysteinemic neurons. Further analysis revealed that tau network proteins played functional roles in homocysteine‑induced neuronal damage.

  8. Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

    Science.gov (United States)

    Chen, Yu-Han; Yeh, Ting-Feng; Chu, Fang-Hua; Hsu, Fu-Lan; Chang, Shang-Tzen

    2015-01-14

    Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton β-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)–multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

  9. Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typimurium in response to infection-like conditions

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Wu, Si; Meng, Da; Liu, Xiaowen; Brewer, Heather M.; Kaiser, Brooke LD; Nakayasu, Ernesto S.; Cort, John R.; Pevzner, Pavel A.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; Pasa-Tolic, Ljiljana

    2013-06-18

    Characterization of the mature protein complement in cells is crucial for a better understanding of cellular processes on a systems-wide scale. Bottom-up proteomic approaches often lead to loss of critical information about an endogenous protein’s actual state due to post translational modifications (PTMs) and other processes. Top-down approaches that involve analysis of the intact protein can address this concern but present significant analytical challenges related to the separation quality needed, measurement sensitivity, and speed that result in low throughput and limited coverage. Here we used single-dimension ultra high pressure liquid chromatography mass spectrometry to investigate the comprehensive ‘intact’ proteome of the Gram negative bacterial pathogen Salmonella Typhimurium. Top-down proteomics analysis revealed 563 unique proteins including 1665 proteoforms generated by PTMs, representing the largest microbial top-down dataset reported to date. Our analysis not only confirmed several previously recognized aspects of Salmonella biology and bacterial PTMs in general, but also revealed several novel biological insights. Of particular interest was differential utilization of the protein S-thiolation forms S-glutathionylation and S-cysteinylation in response to infection-like conditions versus basal conditions, which was corroborated by changes in corresponding biosynthetic pathways. This differential utilization highlights underlying metabolic mechanisms that modulate changes in cellular signaling, and represents to our knowledge the first report of S-cysteinylation in Gram negative bacteria. The demonstrated utility of our simple proteome-wide intact protein level measurement strategy for gaining biological insight should promote broader adoption and applications of top-down proteomics approaches.

  10. A Proteomic Approach of Bradyrhizobium/Aeschynomene Root and Stem Symbioses Reveals the Importance of the fixA Locus for Symbiosis

    Directory of Open Access Journals (Sweden)

    Nathanael Delmotte

    2014-02-01

    Full Text Available Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants. Here we used a proteomics approach to investigate the bacterial physiology of photosynthetic Bradyrhizobium sp. ORS278 during the symbiotic process with the semi aquatical plant Aeschynomene indica that forms root and stem nodules. We analyzed the proteomes of bacteria extracted from each type of nodule. First, we analyzed the bacteroid proteome at two different time points and found only minor variation between the bacterial proteomes of 2-week- and 3-week-old nodules. High conservation of the bacteroid proteome was also found when comparing stem nodules and root nodules. Among the stem nodule specific proteins were those related to the phototrophic ability of Bradyrhizobium sp. ORS278. Furthermore, we compared our data with those obtained during an extensive genetic screen previously published. The symbiotic role of four candidate genes which corresponding proteins were found massively produced in the nodules but not identified during this screening was examined. Mutant analysis suggested that in addition to the EtfAB system, the fixA locus is required for symbiotic efficiency.

  11. Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

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    Peter A. Bell

    2013-06-01

    Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

  12. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    Science.gov (United States)

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-05

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

  13. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    Science.gov (United States)

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  14. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

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    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  15. Proteomic Analysis of Fetal Ovaries Reveals That Primordial Follicle Formation and Transition Are Differentially Regulated

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    Mengmeng Xu

    2017-01-01

    Full Text Available Primordial follicle formation represents a critical phase of the initiation of embryonic reproductive organ development, while the primordial follicle transition into primary follicle determines whether oestrus or ovulation will occur in female animals. To identify molecular mechanism of new proteins which are involved in ovarian development, we employed 2D-DIGE to compare the protein expression profiles of primordial follicles and primary follicles of fetal ovaries in pigs. Fetal ovaries were collected at distinct time-points of the gestation cycle (g55 and g90. The identified proteins at the g55 time-point are mainly involved in the development of anatomical structures [reticulocalbin-1 (RCN1, reticulocalbin-3 (RCN3], cell differentiation (actin, and stress response [heterogeneous nuclear ribonucleoprotein K (HNRNPK]. Meanwhile, at the g90 stage, the isolated proteins with altered expression levels were mainly associated with cell proliferation [major vault protein (MVP] and stress response [heat shock-related 70 kDa protein 2 (HSPA2]. In conclusion, our work revealed that primordial follicle formation is regulated by RCN1, RCN3, actin, and HNRNPK, while the primordial follicle transformation to primary follicle is regulated by MVP and HSPA2. Therefore, our results provide further information for the prospective understanding of the molecular mechanism(s involved in the regulation of the ovarian follicle development.

  16. Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses.

    Science.gov (United States)

    You, Xiangrong; Wang, Lingxia; Liang, Wenyu; Gai, Yonghong; Wang, Xiaoyan; Chen, Wei

    2012-02-02

    Two-dimensional gel electrophoresis (2-DE) was used to analyze the proteins related to floral reversion in Dimocarpus longan Lour. Proteins were extracted from buds undergoing the normal process of flowering and from those undergoing floral reversion in three developing stages in D. longan. Differentially expressed proteins were identified from the gels after 2-DE analysis, which were confirmed using matrix-assisted laser desorption/ionization-time of flying-mass spectroscopy and protein database search. A total of 39 proteins, including 18 up-regulated and 21 down-regulated proteins, were classified into different categories, such as energy and substance metabolism, protein translation, secondary metabolism, phytohormone, cytoskeleton structure, regulation, and stress tolerance. Among these, the largest functional class was associated with primary metabolism. Down-regulated proteins were involved in photosynthesis, transcription, and translation, whereas up-regulated proteins were involved in respiration. Decreased flavonoid synthesis and up-regulated GA20ox might be involved in the floral reversion process. Up-regulated 14-3-3 proteins played a role in the regulation of floral reversion in D. longan by responding to abiotic stress. Observations via transmission electron microscopy revealed the ultrastructure changes in shedding buds undergoing floral reversion. Overall, the results provided insights into the molecular basis for the floral reversion mechanism in D. longan. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

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    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  18. Neutrophil proteomic analysis reveals the participation of antioxidant enzymes, motility and ribosomal proteins in the prevention of ischemic effects by preconditioning

    DEFF Research Database (Denmark)

    Arshid, S; Tahir, M; Fontes, B

    2017-01-01

    therapeutic application; however the exact underlying mechanism is not clear. Neutrophils play an important role in the mechanism of injuries caused by ischemia and reperfusion while IPC led to a decrease in neutrophil stimulation and activation. The effect of preconditioning on the neutrophil proteome...... in such conditions, there is no report of a proteomic study providing a broader view of this scenario. Here we describe a group of proteins significantly regulated by ischemia and reperfusion being such regulation prevented by preconditioning. Such finding may provide relevant information for a deeper understanding...

  19. Proteomic study of benign and malignant pleural effusion.

    Science.gov (United States)

    Li, Hongqing; Tang, Zhonghao; Zhu, Huili; Ge, Haiyan; Cui, Shilei; Jiang, Weiping

    2016-06-01

    Lung adenocarcinoma can easily cause malignant pleural effusion which was difficult to discriminate from benign pleural effusion. Now there was no biomarker with high sensitivity and specificity for the malignant pleural effusion. This study used proteomics technology to acquire and analyze the protein profiles of the benign and malignant pleural effusion, to seek useful protein biomarkers with diagnostic value and to establish the diagnostic model. We chose the weak cationic-exchanger magnetic bead (WCX-MB) to purify peptides in the pleural effusion, used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain peptide expression profiles from the benign and malignant pleural effusion samples, established and validated the diagnostic model through a genetic algorithm (GA) and finally identified the most promising protein biomarker. A GA diagnostic model was established with spectra of 3930.9 and 2942.8 m/z in the training set including 25 malignant pleural effusion and 26 benign pleural effusion samples, yielding both 100 % sensitivity and 100 % specificity. The accuracy of diagnostic prediction was validated in the independent testing set with 58 malignant pleural effusion and 34 benign pleural effusion samples. Blind evaluation was as follows: the sensitivity was 89.6 %, specificity 88.2 %, PPV 92.8 %, NPV 83.3 % and accuracy 89.1 % in the independent testing set. The most promising peptide biomarker was identified successfully: Isoform 1 of caspase recruitment domain-containing protein 9 (CARD9), with 3930.9 m/z, was decreased in the malignant pleural effusion. This model is suitable to discriminate benign and malignant pleural effusion and CARD9 can be used as a new peptide biomarker.

  20. Cerebrospinal Fluid Proteomics Reveals Potential Pathogenic Changes in the Brains of SIV-infected Monkeys

    OpenAIRE

    Pendyala, Gurudutt; Trauger, Sunia A.; Kalisiak, Ewa; Ellis, Ronald J.; Siuzdak, Gary; Fox, Howard S.

    2009-01-01

    The HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of anti-retroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pathology, we studied the cerebrospinal fluid in a nonhuman primate model for HIV-1-associated neurocognitive disorder. Here we present a simple and efficient liquid chromatograph...

  1. Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes.

    Directory of Open Access Journals (Sweden)

    Jessica Ingram

    2011-09-01

    Full Text Available Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.

  2. iTRAQ reveals proteomic changes during intestine regeneration in the sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Sun, Lina; Xu, Dongxue; Xu, Qinzeng; Sun, Jingchun; Xing, Lili; Zhang, Libin; Yang, Hongsheng

    2017-06-01

    Sea cucumbers have a striking capacity to regenerate most of their viscera after evisceration, which has drawn the interest of many researchers. In this study, the isobaric tag for relative and absolute quantitation (iTRAQ) was utilized to investigate protein abundance changes during intestine regeneration in sea cucumbers. A total of 4073 proteins were identified, and 2321 proteins exhibited significantly differential expressions, with 1100 upregulated and 1221 downregulated proteins. Our results suggest that intestine regeneration constitutes a complex life activity regulated by the cooperation of various biological processes, including cytoskeletal changes, extracellular matrix (ECM) remodeling and ECM-receptor interactions, protein synthesis, signal recognition and transduction, energy production and conversion, and substance transport and metabolism. Additionally, real-time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Our results provided a basis for studying the regulatory mechanisms associated with sea cucumber regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease*

    Science.gov (United States)

    Dewhurst, Henry; Sundararaman, Niveda

    2016-01-01

    Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable

  4. Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease.

    Science.gov (United States)

    Torres, Matthew P; Dewhurst, Henry; Sundararaman, Niveda

    2016-11-01

    Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable

  5. Aspergillus fumigatus spore proteomics and genetics reveal that VeA represses DefA-mediated DNA damage response.

    Science.gov (United States)

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young; Heo, In-Beom; Kim, Young Hwan; Yu, Jae-Hyuk

    2016-10-04

    Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid*

    Science.gov (United States)

    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51–64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic. PMID:26499837

  7. Serum proteomic analysis reveals potential serum biomarkers for occupational medicamentosa-like dermatitis caused by trichloroethylene.

    Science.gov (United States)

    Huang, Peiwu; Ren, Xiaohu; Huang, Zhijun; Yang, Xifei; Hong, Wenxu; Zhang, Yanfang; Zhang, Hang; Liu, Wei; Huang, Haiyan; Huang, Xinfeng; Wu, Desheng; Yang, Linqing; Tang, Haiyan; Zhou, Li; Li, Xuan; Liu, Jianjun

    2014-08-17

    Trichloroethylene (TCE) is an industrial solvent with widespread occupational exposure and also a major environmental contaminant. Occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) is an autoimmune disease and it has become one major hazard in China. In this study, sera from 3 healthy controls and 3 OMLDT patients at different disease stages were used for a screening study by 2D-DIGE and MALDI-TOF-MS/MS. Eight proteins including transthyretin (TTR), retinol binding protein 4 (RBP4), haptoglobin, clusterin, serum amyloid A protein (SAA), apolipoprotein A-I, apolipoprotein C-III and apolipoprotein C-II were found to be significantly altered among the healthy, acute-stage, healing-stage and healed-stage groups. Specifically, the altered expression of TTR, RBP4 and haptoglobin were further validated by Western blot analysis and ELISA. Our data not only suggested that TTR, RBP4 and haptoglobin could serve as potential serum biomarkers of OMLDT, but also indicated that measurement of TTR, RBP4 and haptoglobin or their combination could help aid in the diagnosis, monitoring the progression and therapy of the disease. Copyright © 2014. Published by Elsevier Ireland Ltd.

  8. Differential proteomic analysis of Aspergillus fumigatus morphotypes reveals putative drug targets.

    Science.gov (United States)

    Kubitschek-Barreira, Paula H; Curty, Nathalia; Neves, Gabriela W P; Gil, Concha; Lopes-Bezerra, Leila M

    2013-01-14

    Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, an important opportunistic infection for neutropenic patients. The main risk groups are patients with acute leukemia and bone marrow transplantation recipients. The lack of an early diagnostic test together with the limited spectrum of antifungal drugs remains a setback to the successful treatment of this disease. During invasive infection the inhaled fungal conidia enter the morphogenic cycle leading to angioinvasive hyphae. This work aimed to study differentially expressed proteins of A. fumigatus during morphogenesis. To achieve this goal, a 2D-DIGE approach was applied to study surface proteins extractable by reducing agents of two A. fumigatus morphotypes: germlings and hyphae. Sixty-three differentially expressed proteins were identified by MALDI-ToF/MS. We observed that proteins associated with biosynthetic pathways and proteins with multiple functions (miscellaneous) were over-expressed in the early stages of germination, while in hyphae, the most abundant proteins detected were related to metabolic processes or have unknown functions. Among the most interesting proteins regulated during morphogenesis, two putative drug targets were identified, the translational factor, eEF3 and the CipC-like protein. Neither of these proteins are present in mammalian cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

    DEFF Research Database (Denmark)

    Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm

    2011-01-01

    Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain Bifidobacte......Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain...... Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...... functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues...

  10. Final Report: Proteomic study of brassinosteroid responses in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhiyong [Carnegie Inst. of Washington, Argonne, IL (United States); Burlingame, Alma [Univ. of California, San Francisco, CA (United States)

    2017-11-29

    The steroid hormone brassinosteroid (BR) is a major growth-promoting phytohormone. The specific aim of the current project is to identify BR-regulated proteins and characterize their functions in various aspects of plant growth, development, and adaptation. Our research has significantly advanced our understanding of how BR signal is transduced from the receptor at the cell surface to changes of nuclear gene expression and other cellular responses such as vesicle trafficking, as well as developmental transitions such as seed germination and flowering. We have also developed effective proteomic methods for quantitative analysis of protein phosphorylation and for identification of glycosylated proteins. Through this DOE funding, we have performed several proteomic experiments and made major discoveries.

  11. Semen proteomics and male infertility.

    Science.gov (United States)

    Jodar, Meritxell; Soler-Ventura, Ada; Oliva, Rafael

    2017-06-06

    Semen is a complex body fluid containing an admixture of spermatozoa suspended in secretions from the testes and epididymis which are mixed at the time of ejaculation with secretions from other accessory sex glands such as the prostate and seminal vesicles. High-throughput technologies have revealed that, contrary to the idea that sperm cells are simply a silent delivery vehicle of the male genome to the oocyte, the sperm cells in fact provide both a specific epigenetically marked DNA together with a complex population of proteins and RNAs crucial for embryogenesis. Similarly, -omic technologies have also enlightened that seminal fluid seems to play a much greater role than simply being a medium to carry the spermatozoa through the female reproductive tract. In the present review, we briefly overview the sperm cell biology, consider the key issues in sperm and seminal fluid sample preparation for high-throughput proteomic studies, describe the current state of the sperm and seminal fluid proteomes generated by high-throughput proteomic technologies and provide new insights into the potential communication between sperm and seminal fluid. In addition, comparative proteomic studies open a window to explore the potential pathogenic mechanisms of infertility and the discovery of potential biomarkers with clinical significance. The review updates the numerous proteomics studies performed on semen, including spermatozoa and seminal fluid. In addition, an integrative analysis of the testes, sperm and seminal fluid proteomes is also included providing insights into the molecular mechanisms that regulate the generation, maturation and transit of spermatozoa. Furthermore, the compilation of several differential proteomic studies focused on male infertility reveals potential pathways disturbed in specific subtypes of male infertility and points out towards future research directions in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

    Science.gov (United States)

    Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

    2013-01-14

    The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Proteomic Prediction of Breast Cancer Risk: A Cohort Study

    Science.gov (United States)

    2009-03-01

    the SCX equilibrate-system program under Method, to bring all solutions to running- condition. ( Important ! Manually purge both pumps in each delivery...affinity tags. Nat Biotechnol. 1999;17:994- 999.[PMID: 10504701] 10. Washburn MP, Wolters D, Yates JR 3rd. Large-scale analysis of the yeast proteome by...X, Wang R, Wasinger V, Wu CY, Zhao X, Zeng R, Archakov A, Tsugita A, Beer I, Pandey A, Pisano M, Andrews P, Tammen H, Speicher DW, Hanash SM

  14. Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

    Science.gov (United States)

    Hu, Xiaolong; Zhu, Min; Liang, Zi; Kumar, Dhiraj; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2017-04-01

    The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

  15. Quantitative Proteomics Reveals Ecophysiological Effects of Light and Silver Stress on the Mixotrophic Protist Poterioochromonas malhamensis.

    Directory of Open Access Journals (Sweden)

    Daniela Beisser

    Full Text Available Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to

  16. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

    Science.gov (United States)

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

    2016-02-18

    in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls. Our novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development.

  17. Proteomic study related to vascular connections in watermelon scions grafted onto bottle-gourd rootstock under different light intensities.

    Directory of Open Access Journals (Sweden)

    Sowbiya Muneer

    Full Text Available Although grafting is broadly used in the production of crops, no information is available about the proteins involved in vascular connections between rootstock and scion. Similarly, proteome changes under the light intensities widely used for grafted seedlings are of practical use. The objective of this study was to determine the proteome of vascular connections using watermelon (Citrullus vulgaris Schrad. 'Sambok Honey' and 'Speed' as the scion and bottle gourd (Lagenaria siceraria Stanld. 'RS Dongjanggun' as the rootstock grown under different light intensities (25, 50, 75 and 100 μmol m-2 s-1. Our proteomic analysis revealed 24 and 27 differentially expressed proteins in 'Sambok Honey' and 'Speed', respectively, under different light intensities. The identified proteins were largely involved in ion binding, amino acid metabolism, transcriptional regulation and defense response. The enhancement of ion-binding, transcriptional regulation, amino acid metabolism, and defense response proteins suggests a strengthening of the connection between the rootstock and scion under high light intensity. Indeed, the accumulation of key enzymes in the biological processes described above appears to play an important role in the vascular connections of grafted seedlings. Moreover, it appears that 100 μmol m-2 s-1 results in better protein expression responses in grafted seedlings.

  18. Maintenance of neural activities in torpid Rhinolophus ferrumequinum bats revealed by 2D gel-based proteome analysis.

    Science.gov (United States)

    Yin, Qiuyuan; Zhang, Yijian; Dong, Dong; Lei, Ming; Zhang, Shuyi; Liao, Chen-Chung; Pan, Yi-Hsuan

    2017-08-01

    Bats are the only mammals capable of self-powered flying. Many bat species hibernate in winter. A reversible control of cerebral activities is critical for bats to accommodate a repeated torpor-arousal cycle during hibernation. Little is known about the molecular mechanisms that regulate neuronal activities in torpid bats. In this study, Rhinolophus ferrumequinum bat brain proteins were fractionated, and their abundance in active and torpid states was compared. Results of 2D gel-based proteomics showed that 38% of identified proteins with a significant change in abundance are involved in synaptic vesicle recycling and cytoskeletal integrity. Changes in the abundance of proteins related to RNA splicing, proteostasis, redox homeostasis, mitochondrial function, and energy metabolism were also detected. In addition, the levels of GNAO1 (guanine nucleotide-binding protein G αo subunit), an important modulator of neuronal transmembrane signaling, were significantly increased in the insoluble protein fraction of torpid bats; this may be due to GNAO1 palmitoylation making it insoluble. Our data provide molecular evidence for the maintenance of neuronal activities in torpid bats and suggest that a reversible palmitoylation of the G protein plays a role in the regulation of neuronal activities during bat hibernation. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Proteome analysis reveals distinct uranium stress response in two strains of Cyanobacteria native to Indian paddy fields

    International Nuclear Information System (INIS)

    Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar

    2017-01-01

    Uranium present in phosphate fertilizer contaminates agricultural land. Uranium exerts chemical toxicity to the resident biota as it induces oxidative stress by generating free radicals. Two strains of nitrogen fixing cyanobacteria viz., Anabaena PCC 7120 and L-31 native to Indian paddy, regularly experience oxidative stress induced by different stresses and heavy metals. The present study investigated their response to uranium exposure at proteomic level. LD_5_0 dose for Anabaena 7120 and Anabaena L-31 was determined to be 75 μM and 200 μM uranyl carbonate exposure for 3 h. A total of 79 proteins from Anabaena 7120 and 64 proteins from Anabaena L-31 were identified by MALDI mass spectrometry, of which levels of 45 and 27 proteins respectively were found to be differentially modulated in the two strains in response to uranium exposure. The differentially expressed proteins belonged to the major functional categories of photosynthesis, carbon metabolism and oxidative stress alleviation, commensurate with their uranium tolerance. Better oxidative stress management, and maintenance of metabolic and energy homeostasis lead to superior uranium tolerance in Anabaena L-31 as compared to Anabaena PCC 7120

  20. iTRAQ proteomics analysis reveals that PI3K is highly associated with bupivacaine-induced neurotoxicity pathways.

    Science.gov (United States)

    Zhao, Wei; Liu, Zhongjie; Yu, Xujiao; Lai, Luying; Li, Haobo; Liu, Zipeng; Li, Le; Jiang, Shan; Xia, Zhengyuan; Xu, Shi-yuan

    2016-02-01

    Bupivacaine, a commonly used local anesthetic, has potential neurotoxicity through diverse signaling pathways. However, the key mechanism of bupivacaine-induced neurotoxicity remains unclear. Cultured human SH-SY5Y neuroblastoma cells were treated (bupivacaine) or untreated (control) with bupivacaine for 24 h. Compared to the control group, bupivacaine significantly increased cyto-inhibition, cellular reactive oxygen species, DNA damage, mitochondrial injury, apoptosis (increased TUNEL-positive cells, cleaved caspase 3, and Bcl-2/Bax), and activated autophagy (enhanced LC3II/LC3I ratio). To explore changes in protein expression and intercommunication among the pathways involved in bupivacaine-induced neurotoxicity, an 8-plex iTRAQ proteomic technique and bioinformatics analysis were performed. Compared to the control group, 241 differentially expressed proteins were identified, of which, 145 were up-regulated and 96 were down-regulated. Bioinformatics analysis of the cross-talk between the significant proteins with altered expression in bupivacaine-induced neurotoxicity indicated that phosphatidyl-3-kinase (PI3K) was the most frequently targeted protein in each of the interactions. We further confirmed these results by determining the downstream targets of the identified signaling pathways (PI3K, Akt, FoxO1, Erk, and JNK). In conclusion, our study demonstrated that PI3K may play a central role in contacting and regulating the signaling pathways that contribute to bupivacaine-induced neurotoxicity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

    Science.gov (United States)

    Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

    2013-04-01

    Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide

    DEFF Research Database (Denmark)

    Cassoli, Juliana Silva; Verano-Braga, Thiago; Oliveira, Joacir Stolarz

    2013-01-01

    duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low...... present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy...

  3. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

    2005-01-01

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  4. A proteomics study of in vitro cyst germination and appressoria formation in ¤Phytophthora infestans¤

    DEFF Research Database (Denmark)

    Ebstrup, T.; Saalbach, G.; Egsgaard, H.

    2005-01-01

    A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from ...... five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots...

  5. Revealing critical mechanisms of BR-mediated apple nursery tree growth using iTRAQ-based proteomic analysis.

    Science.gov (United States)

    Zheng, Liwei; Ma, Juanjuan; Zhang, Lizhi; Gao, Cai; Zhang, Dong; Zhao, Caiping; Han, Mingyu

    2018-02-20

    Brassinosteroid is identified as an important hormone. However, information about brassinosteroid has not been fully elucidated, and few studies concerned its role in apple. The aim of this work was to study the role of brassinosteroid for apple tree growth. In our study, the effect of brassinosteroid on apple nursery tree was analyzed. The biomass, cell size and xylem content of apple nursery tree were obviously evaluated by brassinosteroid treatment; mineral elements contents, photosynthesis indexes, carbohydrate level and hormone contents were significantly high in brassinosteroid treated trees. To explore the molecular mechanisms of these phenotypic differences, iTRAQ-based quantitative proteomics were used to identify the expression profiles of proteins in apple nursery tree shoot tips in response to brassinosteroid at a key period (14days after brassinosteroid treatment). A total of 175 differentially expressed proteins were identified. They were mainly involved in chlorophyII biosynthesis, photosynthesis, carbohydrate metabolism, glycolysis, citric acid cycle, respiratory action, hormone signal, cell growth and ligin metabolism. The findings in this study indicate that brassinosteroid mediating apple nursery tree growth may be mainly through energy metabolism. Important biological processes identified here can be useful theoretical basis and provide new insights into the molecular mechanisms of brassinosteroid. Brassinosteroid is very important for plant growth and development. However, the molecular mechanism of brassinosteroid mediating growth process is not perfectly clear in plant, especially in apple nursery tree. We used a combination of physiological and bioinformatics analysis to investigate the effects of brassinosteroid on apple nursery tree growth and development. The data reported here demonstrated that brassinosteroid regulates apple nursery tree growth mainly through energy metabolism. Therefore it can provide a theoretical basis from energy

  6. SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus

    Science.gov (United States)

    Li, Bing; Tian, Xiaofang; Wang, Chunlan; Zeng, Xu; Xing, Yongmei; Ling, Hong; Yin, Wanqiang; Tian, Lixia; Meng, Zhixia; Zhang, Jihui; Guo, Shunxing

    2017-01-01

    Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.

  7. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    KAUST Repository

    An, Xiaomeng; Shao, Jiaofang; Zhang, Huoming; Ren, Xiaoliang; Ho, Vincy Wing Sze; Li, Runsheng; Wong, Ming-Kin; Zhao, Zhongying

    2017-01-01

    . The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins

  8. Proteomics of the rat myocardium during development of type 2 diabetes mellitus reveals progressive alterations in major metabolic pathways

    DEFF Research Database (Denmark)

    Edhager, Anders Valdemar; Povlsen, Jonas Agerlund; Løfgren, Bo

    2018-01-01

    in intracellular metabolic pathways in the Zucker diabetic fatty rat heart as T2DM develops using MS based proteomics. The pre-diabetic state only induced minor pathway changes, whereas onset and late T2DM caused pronounced perturbations. Two actin-associated proteins, ARPC2 and TPM3, were up-regulated at the pre...

  9. Comparative proteomics of oxidative stress response of Lactobacillus acidophilus NCFM reveals effects on DNA repair and cysteine de novo synthesis

    DEFF Research Database (Denmark)

    Calderini, Elia; Celebioglu, Hasan Ufuk; Villarroel, Julia

    2017-01-01

    acidophilus NCFM to H2O2, simulating an oxidative environment. Bacterial growth was monitored by BioScreen and batch cultures were harvested at exponential phase for protein profiling of stress responses by 2D gel-based comparative proteomics. Proteins identified in 19 of 21 spots changing in abundance due...

  10. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Kwang-Soo, E-mail: shinks@dju.kr [Division of Life Science, Daejeon University, Daejeon, 300-716 (Korea, Republic of); Kim, Young Hwan [Biomedical Omics Team, Korea Basic Science Institute (KBSI), Ohcang, 368-883 (Korea, Republic of); Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 305-333 (Korea, Republic of); Yu, Jae-Hyuk, E-mail: jyu1@wisc.edu [Departments of Bacteriology and Genetics, The University of Wisconsin–Madison, Madison, WI, 53706 (United States)

    2015-07-31

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.

  11. Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

    International Nuclear Information System (INIS)

    Shin, Kwang-Soo; Kim, Young Hwan; Yu, Jae-Hyuk

    2015-01-01

    The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus

  12. Proteomic Analysis of Interaction between a Plant Virus and Its Vector Insect Reveals New Functions of Hemipteran Cuticular Protein.

    Science.gov (United States)

    Liu, Wenwen; Gray, Stewart; Huo, Yan; Li, Li; Wei, Taiyun; Wang, Xifeng

    2015-08-01

    Numerous viruses can be transmitted by their corresponding vector insects; however, the molecular mechanisms enabling virus transmission by vector insects have been poorly understood, especially the identity of vector components interacting with the virus. Here, we used the yeast two-hybrid system to study proteomic interactions of a plant virus (Rice stripe virus, RSV, genus Tenuivirus) with its vector insect, small brown planthopper (Laodelphax striatellus). Sixty-six proteins of L. striatellus that interacted with the nucleocapsid protein (pc3) of RSV were identified. A virus-insect interaction network, constructed for pc3 and 29 protein homologs of Drosophila melanogaster, suggested that nine proteins might directly interact with pc3. Of the 66 proteins, five (atlasin, a novel cuticular protein, jagunal, NAC domain protein, and vitellogenin) were most likely to be involved in viral movement, replication, and transovarial transmission. This work also provides evidence that the novel cuticular protein, CPR1, from L. striatellus is essential for RSV transmission by its vector insect. CPR1 binds the nucleocapsid protein (pc3) of RSV both in vivo and in vitro and colocalizes with RSV in the hemocytes of L. striatellus. Knockdown of CPR1 transcription using RNA interference resulted in a decrease in the concentration of RSV in the hemolymph, salivary glands and in viral transmission efficiency. These data suggest that CPR1 binds RSV in the insect and stabilizes the viral concentration in the hemolymph, perhaps to protect the virus or to help move the virus to the salivary tissues. Our studies provide direct experimental evidence that viruses can use existing vector proteins to aid their survival in the hemolymph. Identifying these putative vector molecules should lead to a better understanding of the interactions between viruses and vector insects. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Qun Liu

    2012-01-01

    Full Text Available Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale. In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

  14. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

    Science.gov (United States)

    Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

    2017-01-01

    Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

  15. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

    Directory of Open Access Journals (Sweden)

    Dominik A. Megger

    2017-09-01

    Full Text Available Interferons (IFNs are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction. In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

  16. [Proteomics and transfusion medicine].

    Science.gov (United States)

    Lion, N; Prudent, M; Crettaz, D; Tissot, J-D

    2011-04-01

    The term "proteomics" covers tools and techniques that are used to analyze and characterize complex mixtures of proteins from various biological samples. In this short review, a typical proteomic approach, related to the study of particular and illustrative situation related to transfusion medicine is reported. This "case report" will allow the reader to be familiar with a practical proteomic approach of a real situation, and will permit to describe the tools that are usually used in proteomic labs, and, in a second part, to present various proteomic applications in transfusion medicine. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Analysis of the embryo proteome of sycamore (Acer pseudoplatanus L.) seeds reveals a distinct class of proteins regulating dormancy release.

    Science.gov (United States)

    Pawłowski, Tomasz Andrzej; Staszak, Aleksandra Maria

    2016-05-20

    Acer pseudoplatanus seeds are characterized by a deep physiological embryo dormancy that requires a few weeks of cold stratification in order to promote germination. Understanding the function of proteins and their related metabolic pathways, in conjunction with the plant hormones implicated in the breaking of seed dormancy, would expand our knowledge pertaining to this process. In this study, a proteomic approach was used to analyze the changes occurring in seeds in response to cold stratification, which leads to dormancy release. In addition, the involvement of abscisic (ABA) and gibberellic acids (GA) was also examined. Fifty-three proteins showing significant changes were identified by mass spectrometry. An effect of ABA on protein variation was observed at the beginning of stratification, while the influence of GA on protein abundance was observed during the middle phase of stratification. The majority of proteins associated with dormancy breaking in the presence of only water, and also ABA or GA, were classified as being involved in metabolism and genetic information processing. For metabolic-related proteins, the effect of ABA on protein abundance was stimulatory for half of the proteins and inhibitory for half of the proteins. On the other hand, the effect on genetic information processing related proteins was stimulatory. GA was found to upregulate both metabolic-related and genetic information processing-related proteins. While seed dormancy breaking depends on proteins involved in a variety of processes, proteins associated with methionine metabolism (adenosine kinase, methionine synthase) and glycine-rich RNA binding proteins appear to be of particular importance. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. Quantitative proteomics reveals divergent responses in Apis mellifera worker and drone pupae to parasitization by Varroa destructor.

    Science.gov (United States)

    Surlis, Carla; Carolan, James C; Coffey, Mary; Kavanagh, Kevin

    Varroa destructor is a haemophagous ectoparasite of honeybees and is considered a major causal agent of colony losses in Europe and North America. Although originating in Eastern Asia where it parasitizes Apis cerana, it has shifted hosts to the western honeybee Apis mellifera on which it has a greater deleterious effect on the individual and colony level. To investigate this important host-parasite interaction and to determine whether Varroa causes different effects on different castes we conducted a label free quantitative proteomic analysis of Varroa-parasitized and non-parasitized drone and worker Apis mellifera pupae. 1195 proteins were identified in total, of which 202 and 250 were differentially abundant in parasitized drone and worker pupae, respectively. Both parasitized drone and worker pupae displayed reduced abundance in proteins associated with the cuticle, lipid transport and innate immunity. Proteins involved in metabolic processes were more abundant in both parasitized castes although the response in workers was more pronounced. A number of caste specific responses were observed including differential abundance of numerous cytoskeletal and muscle proteins, which were of higher abundance in parasitized drones in comparison to parasitized workers. Proteins involved in fatty acid and carbohydrate metabolism were more abundant in parasitized workers as were a large number of ribosomal proteins highlighting either potentially divergent responses to Varroa or a different strategy by the mite when parasitizing the different castes. This data improves our understanding of this interaction and may provide a basis for future studies into improvements to therapy and control of Varroasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

    Science.gov (United States)

    Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

    2017-01-01

    Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

  20. Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli; Sun, Jin; Mok, FloraSy; Liu, Lingli; Qiu, Jianwen; Ravasi, Timothy; Qian, Peiyuan

    2013-01-01

    Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

  1. Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2013-03-01

    Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

  2. Comparative proteomic analysis of lung tissue from guinea pigs with Leptospiral Pulmonary Haemorrhage Syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

    Science.gov (United States)

    The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, a 2-D guinea pig proteome lung map was used to investigate the pathogenic mechanisms of ...

  3. Proteomic analysis of grapevine resistance induced by Trichoderma harzianum T39 reveals specific defence pathways activated against downy mildew

    Science.gov (United States)

    Perazzolli, Michele

    2012-01-01

    Downy mildew is caused by the oomycete Plasmopara viticola and is one of the most serious diseases of grapevine. The beneficial microorganism Trichoderma harzianum T39 (T39) has previously been shown to induce plant-mediated resistance and to reduce the severity of downy mildew in susceptible grapevines. In order to better understand the cellular processes associated with T39-induced resistance, the proteomic and histochemical changes activated by T39 in grapevine were investigated before and 1 day after P. viticola inoculation. A comprehensive proteomic analysis of T39-induced resistance in grapevine was performed using an eight-plex iTRAQ protocol, resulting in the identification and quantification of a total of 800 proteins. Most of the proteins directly affected by T39 were found to be involved in signal transduction, indicating activation of a complete microbial recognition machinery. Moreover, T39-induced resistance was associated with rapid accumulation of reactive oxygen species and callose at infection sites, as well as changes in abundance of proteins involved in response to stress and redox balance, indicating an active defence response to downy mildew. On the other hand, proteins affected by P. viticola in control plants mainly decreased in abundance, possibly reflecting the establishment of a compatible interaction. Finally, the high-throughput iTRAQ protocol allowed de novo peptide sequencing, which will be used to improve annotation of the Vitis vinifera cv. Pinot Noir proteome. PMID:23105132

  4. The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis.

    Science.gov (United States)

    Yu, Yadong; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

    2017-03-07

    Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.

  5. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

    NARCIS (Netherlands)

    Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.; Acha, Moshe Ray; Newton-Cheh, Christopher; Pfeufer, Arne; Lyneh, Stacey N.; Olesen, Soren-Peter; Brunak, Soren; Ellinor, Patrick T.; Jukema, J. Wouter; Trompet, Stella; Ford, Ian; Macfarlane, Peter W.; Krijthe, Bouwe P.; Hofman, Albert; Uitterlinden, Andre G.; Stricker, Bruno H.; Nathoe, Hendrik M.; Spiering, Wilko; Daly, Mark J.; Asselbergs, Ikea W.; van der Harst, Pim; Milan, David J.; de Bakker, Paul I. W.; Lage, Kasper; Olsen, Jesper V.

    Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes

  6. Proteomic study on the stability of proteins in bovine, camel, and caprine milk sera after processing

    NARCIS (Netherlands)

    Zhang, Lina; Boeren, Sjef; Smits, Marcel; Hooijdonk, van Toon; Vervoort, Jacques; Hettinga, Kasper

    2016-01-01

    Milk proteins have been shown to be very sensitive to processing. This study aims to investigate the changes of the bovine, camel, and caprine milk proteins after freezing, pasteurization (62 °C, 30 min), and spray drying by proteomic techniques, filter-aided sample preparation (FASP) and

  7. Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs.

    Directory of Open Access Journals (Sweden)

    Mengmeng Xu

    Full Text Available Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins and molecular functions (enzyme regulator activity. We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be

  8. Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs.

    Science.gov (United States)

    Xu, Mengmeng; Che, Long; Wang, Dingyue; Yang, Zhenguo; Zhang, Pan; Lin, Yan; Fang, Zhengfeng; Che, Lianqiang; Li, Jian; Chen, Daiwen; Wu, De; Xu, Shengyu

    2015-01-01

    Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for

  9. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

    Directory of Open Access Journals (Sweden)

    Kristin eSurmann

    2014-08-01

    Full Text Available Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549, and human embryonic kidney cells (HEK 293. Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen´s proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2x106 bacteria, roughly 1,450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreases in levels of ribosomal proteins and metabolic enzymes or increases in amounts of proteins involved in arginine and lysine biosynthesis, coding for terminal oxidases and stress responsive genes or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and

  10. The core proteome and pan proteome of Salmonella Paratyphi A epidemic strains.

    Directory of Open Access Journals (Sweden)

    Li Zhang

    Full Text Available Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

  11. Bacterial Survival under Extreme UV Radiation: A Comparative Proteomics Study of Rhodobacter sp., Isolated from High Altitude Wetlands in Chile

    Directory of Open Access Journals (Sweden)

    Vilma Pérez

    2017-06-01

    Full Text Available Salar de Huasco, defined as a polyextreme environment, is a high altitude saline wetland in the Chilean Altiplano (3800 m.a.s.l., permanently exposed to the highest solar radiation doses registered in the world. We present here the first comparative proteomics study of a photoheterotrophic bacterium, Rhodobacter sp., isolated from this remote and hostile habitat. We developed an innovative experimental approach using different sources of radiation (in situ sunlight and UVB lamps, cut-off filters (Mylar, Lee filters and a high-throughput, label-free quantitative proteomics method to comprehensively analyze the effect of seven spectral bands on protein regulation. A hierarchical cluster analysis of 40 common proteins revealed that all conditions containing the most damaging UVB radiation induced similar pattern of protein regulation compared with UVA and visible light spectral bands. Moreover, it appeared that the cellular adaptation of Rhodobacter sp. to osmotic stress encountered in the hypersaline environment from which it was originally isolated, might further a higher resistance to damaging UV radiation. Indeed, proteins involved in the synthesis and transport of key osmoprotectants, such as glycine betaine and inositol, were found in very high abundance under UV radiation compared to the dark control, suggesting the function of osmolytes as efficient reactive oxygen scavengers. Our study also revealed a RecA-independent response and a tightly regulated network of protein quality control involving proteases and chaperones to selectively degrade misfolded and/or damaged proteins.

  12. HUPO BPP pilot study: a proteomics analysis of the mouse brain of different developmental stages.

    Science.gov (United States)

    Wang, Jing; Gu, Yong; Wang, Lihong; Hang, Xingyi; Gao, Yan; Wang, Hangyan; Zhang, Chenggang

    2007-11-01

    This study is a part of the HUPO Brain Proteome Project (BPP) pilot study, which aims at obtaining a reliable database of mouse brain proteome, at the comparison of techniques, laboratories, and approaches as well as at preparing subsequent proteome studies of neurologic diseases. The C57/Bl6 mouse brains of three developmental stages at embryonic day 16 (E16), postnatal day 7 (P7), and 8 wk (P56) (n = 5 in each group) were provided by the HUPO BPP executive committee. The whole brain proteins of each animal were individually prepared using 2-DE coupled with PDQuest software analysis. The protein spots representing developmentally related or stably expressed proteins were then prepared with in-gel digestion followed with MALDI-TOF/TOF MS/MS and analyzed using the MASCOT search engines to search the Swiss-Prot or NCBInr database. The 2-DE gel maps of the mouse brains of all of the developmental stages were obtained and submitted to the Data Collection Centre (DCC). The proteins alpha-enolase, stathmin, actin, C14orf166 homolog, 28,000 kDa heat- and acid-stable phosphoprotein, 3-mercaptopyruvate sulfurtransferase and 40 S ribosomal protein S3a were successfully identified. A further Western blotting analysis demonstrated that enolase is a protein up-regulated in the mouse brain from embryonic stage to adult stage. These data are helpful for understanding the proteome changes in the development of the mouse brain.

  13. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    Directory of Open Access Journals (Sweden)

    Allison Marie Barbaglia

    2016-04-01

    Full Text Available Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho- lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012. Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I a putative GDSL-motif lipase (II a PIG-P-like protein, with a possible receptor-like function; (III and PLAFP (phloem lipid-associated family protein, a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH, which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while

  14. The Seed Proteome Web Portal

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    Marc eGalland

    2012-06-01

    Full Text Available The Seed Proteome Web Portal (SPWP; http://www.seedproteome.com/ gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from 2 dimensional electrophoresis (2DE maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35% or a decreasing abundance (15%. Moreover, during radicle protrusion (24 h to 48 h upon imbibition, 41% proteins display quantitative variations with an increased (23% or a decreasing abundance (18%. In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r2 = 0.29 between the theoretical (predicted from Arabidopsis genome and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthetized proteins radiolabeled by [35S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

  15. IgV peptide mapping of native Ro60 autoantibody proteomes in primary Sjögren's syndrome reveals molecular markers of Ro/La diversification.

    Science.gov (United States)

    Wang, Jing J; Al Kindi, Mahmood A; Colella, Alex D; Dykes, Lukah; Jackson, Michael W; Chataway, Tim K; Reed, Joanne H; Gordon, Tom P

    2016-12-01

    We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

    Science.gov (United States)

    Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

    2017-06-01

    The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

    KAUST Repository

    Schmidt, Angelika

    2018-04-27

    BackgroundRegulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.ResultsTo gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.ConclusionThe data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.

  18. Proteomic Analysis Reveals Coordinated Regulation of Anthocyanin Biosynthesis through Signal Transduction and Sugar Metabolism in Black Rice Leaf.

    Science.gov (United States)

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zheng, Jingui

    2017-12-15

    Black rice ( Oryza sativa L.) is considered to be a healthy food due to its high content of anthocyanins in the pericarp. The synthetic pathway of anthocyanins in black rice grains has been identified, however, the proteomic profile of leaves during grain development is still unclear. Here, isobaric Tags Relative and Absolute Quantification (iTRAQ) MS/MS was carried out to identify statistically significant changes of leaf proteome in the black rice during grain development. Throughout three sequential developmental stages, a total of 3562 proteins were detected and 24 functional proteins were differentially expressed 3-10 days after flowering (DAF). The detected proteins are known to be involved in various biological processes and most of these proteins were related to gene expression regulatory (33.3%), signal transduction (16.7%) and developmental regulation and hormone-like proteins (12.5%). The coordinated changes were consistent with changes in regulatory proteins playing a leading role in leaves during black rice grain development. This indicated that signal transduction between leaves and grains may have an important role in anthocyanin biosynthesis and accumulation during grain development of black rice. In addition, four identified up-regulated proteins associated with starch metabolism suggested that the remobilization of nutrients for starch synthesis plays a potential role in anthocyanin biosynthesis of grain. The mRNA transcription for eight selected proteins was validated with quantitative real-time PCR. Our results explored the proteomics of the coordination between leaf and grain in anthocyanins biosynthesis of grain, which might be regulated by signal transduction and sugar metabolism in black rice leaf.

  19. Proteomic Analysis Reveals Coordinated Regulation of Anthocyanin Biosynthesis through Signal Transduction and Sugar Metabolism in Black Rice Leaf

    Directory of Open Access Journals (Sweden)

    Linghua Chen

    2017-12-01

    Full Text Available Black rice (Oryza sativa L. is considered to be a healthy food due to its high content of anthocyanins in the pericarp. The synthetic pathway of anthocyanins in black rice grains has been identified, however, the proteomic profile of leaves during grain development is still unclear. Here, isobaric Tags Relative and Absolute Quantification (iTRAQ MS/MS was carried out to identify statistically significant changes of leaf proteome in the black rice during grain development. Throughout three sequential developmental stages, a total of 3562 proteins were detected and 24 functional proteins were differentially expressed 3–10 days after flowering (DAF. The detected proteins are known to be involved in various biological processes and most of these proteins were related to gene expression regulatory (33.3%, signal transduction (16.7% and developmental regulation and hormone-like proteins (12.5%. The coordinated changes were consistent with changes in regulatory proteins playing a leading role in leaves during black rice grain development. This indicated that signal transduction between leaves and grains may have an important role in anthocyanin biosynthesis and accumulation during grain development of black rice. In addition, four identified up-regulated proteins associated with starch metabolism suggested that the remobilization of nutrients for starch synthesis plays a potential role in anthocyanin biosynthesis of grain. The mRNA transcription for eight selected proteins was validated with quantitative real-time PCR. Our results explored the proteomics of the coordination between leaf and grain in anthocyanins biosynthesis of grain, which might be regulated by signal transduction and sugar metabolism in black rice leaf.

  20. Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage

    International Nuclear Information System (INIS)

    Bouthier de la Tour, Claire; Passot, Fanny Marie; Toueille, Magali; Servant, Pascale; Sommer, Suzanne; Mirabella, Boris; Blanchard, Laurence; Groot, Arjan de; Guerin, Philippe; Armengaud, Jean

    2013-01-01

    The nucleoids of radiation-resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid-enriched fractions from unirradiated and irradiated Deinococcus were identified and semi quantified by shotgun proteomics. The ssDNA-binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament-like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post-irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196. (authors)

  1. Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes

    NARCIS (Netherlands)

    Revuelta, M.V.; Kan, van J.A.L.; Kay, J.; Have, ten A.

    2014-01-01

    The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the

  2. Proteomics Analysis Reveals Abnormal Electron Transport and Excessive Oxidative Stress Cause Mitochondrial Dysfunction in Placental Tissues of Early-Onset Preeclampsia.

    Science.gov (United States)

    Xu, Zhongwei; Jin, Xiaohan; Cai, Wei; Zhou, Maobin; Shao, Ping; Yang, Zhen; Fu, Rong; Cao, Jin; Liu, Yan; Yu, Fang; Fan, Rong; Zhang, Yan; Zou, Shuang; Zhou, Xin; Yang, Ning; Chen, Xu; Li, Yuming

    2018-04-20

    Early-onset preeclampsia (EOS-PE) refers to preeclampsia that occurred before 34 gestation weeks. This study was conducted to explore the relationship between mitochondrial dysfunction and the pathogenesis of EOS-PE using proteomic strategy. To identify altering expressed mitochondrial proteins between severe EOS-PE and healthy pregnancies, enrichment of mitochondria coupled with iTRAQ-based quantitative proteomic method was performed. IHC and western blot were performed to detect the alteration of changing expression proteins, and confirmed the accuracy of proteomic results. We totally quantified 1372 proteins and screened 132 altering expressed mitochondrial proteins, including 86 down-regulated expression proteins and 46 up-regulated expression proteins (pelectron transport chain and oxidative phosphorylation. Especially, mitochondrial related molecules, PRDX2, PARK7, BNIP3, BCL2, PDHA1, SUCLG1, ACADM and NDUFV1, were involved in energy production process in the matrix and membrane of mitochondria. Our results showed that abnormal electron transport, excessive oxidative stress and mitochondrion disassembly might be the main cause of mitochondrial dysfunction, and was related to the pathogenesis of EOS-PE. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

    Science.gov (United States)

    Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

    2017-07-01

    TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

    Science.gov (United States)

    Wang, Shuzhen; Chen, Wenyue; Xiao, Wenfei; Yang, Changdeng; Xin, Ya; Qiu, Jieren; Hu, Weimin; Ying, Wu; Fu, Yaping; Tong, Jianxin; Hu, Guocheng; Chen, Zhongzhong; Fang, Xianping; Yu, Hong; Lai, Wenguo; Ruan, Songlin; Ma, Huasheng

    2015-01-01

    Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various

  5. Proteomic analysis in type 2 diabetes patients before and after a very low calorie diet reveals potential disease state and intervention specific biomarkers.

    Directory of Open Access Journals (Sweden)

    Maria A Sleddering

    Full Text Available Very low calorie diets (VLCD with and without exercise programs lead to major metabolic improvements in obese type 2 diabetes patients. The mechanisms underlying these improvements have so far not been elucidated fully. To further investigate the mechanisms of a VLCD with or without exercise and to uncover possible biomarkers associated with these interventions, blood samples were collected from 27 obese type 2 diabetes patients before and after a 16-week VLCD (Modifast ∼ 450 kcal/day. Thirteen of these patients followed an exercise program in addition to the VCLD. Plasma was obtained from 27 lean and 27 obese controls as well. Proteomic analysis was performed using mass spectrometry (MS and targeted multiple reaction monitoring (MRM and a large scale isobaric tags for relative and absolute quantitation (iTRAQ approach. After the 16-week VLCD, there was a significant decrease in body weight and HbA1c in all patients, without differences between the two intervention groups. Targeted MRM analysis revealed differences in several proteins, which could be divided in diabetes-associated (fibrinogen, transthyretin, obesity-associated (complement C3, and diet-associated markers (apolipoproteins, especially apolipoprotein A-IV. To further investigate the effects of exercise, large scale iTRAQ analysis was performed. However, no proteins were found showing an exercise effect. Thus, in this study, specific proteins were found to be differentially expressed in type 2 diabetes patients versus controls and before and after a VLCD. These proteins are potential disease state and intervention specific biomarkers.Controlled-Trials.com ISRCTN76920690.

  6. Comparative proteomics reveals the physiological differences between winter tender shoots and spring tender shoots of a novel tea (Camellia sinensis L.) cultivar evergrowing in winter.

    Science.gov (United States)

    Liu, Shengjie; Gao, Jiadong; Chen, Zhongjian; Qiao, Xiaoyan; Huang, Hualin; Cui, Baiyuan; Zhu, Qingfeng; Dai, Zhangyan; Wu, Hualing; Pan, Yayan; Yang, Chengwei; Liu, Jun

    2017-11-20

    A recently discovered tea [Camellia sinensis (L.) O. Kuntze] cultivar can generate tender shoots in winter. We performed comparative proteomics to analyze the differentially accumulated proteins between winter and spring tender shoots of this clonal cultivar to reveal the physiological basis of its evergrowing character during winter. We extracted proteins from the winter and spring tender shoots (newly formed two leaves and a bud) of the evergrowing tea cultivar "Dongcha11" respectively. Thirty-three differentially accumulated high-confidence proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF / TOF MS). Among these, 24 proteins had increased abundance while nine showed were decreased abundance in winter tender shoots as compared with the spring tender shoots. We categorized the differentially accumulated proteins into eight critical biological processes based on protein function annotation including photosynthesis, cell structure, protein synthesis & destination, transporters, metabolism of sugars and polysaccharides, secondary metabolism, disease/defense and proteins with unknown functions. Proteins with increased abundance in winter tender shoots were mainly related to the processes of photosynthesis, cytoskeleton and protein synthesis, whereas those with decreased abundance were correlated to metabolism and the secondary metabolism of polyphenolic flavonoids. Biochemical analysis showed that the total contents of soluble sugar and amino acid were higher in winter tender shoots while tea polyphenols were lower as compared with spring tender shoots. Our study suggested that the simultaneous increase in the abundance of photosynthesis-related proteins rubisco, plastocyanin, and ATP synthase delta chain, metabolism-related proteins eIF4 and protease subunits, and the cytoskeleton-structure associated proteins phosphatidylinositol transfer protein and profilin may be because of the adaptation of the

  7. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials (“aging factors”) through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms. PMID:26351681

  8. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals "aging factors" and mechanism of lifespan asymmetry.

    Science.gov (United States)

    Yang, Jing; McCormick, Mark A; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K; Li, Hao

    2015-09-22

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.

  9. Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

    International Nuclear Information System (INIS)

    Lo, Andy; Weiner, Joel H.; Li, Liang

    2013-01-01

    Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

  10. Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

    Science.gov (United States)

    Delaunois, Bertrand; Jeandet, Philippe; Clément, Christophe; Baillieul, Fabienne; Dorey, Stéphan; Cordelier, Sylvain

    2014-01-01

    Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.

  11. Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy.

    Science.gov (United States)

    McGorum, Bruce C; Pirie, R Scott; Eaton, Samantha L; Keen, John A; Cumyn, Elizabeth M; Arnott, Danielle M; Chen, Wenzhang; Lamont, Douglas J; Graham, Laura C; Llavero Hurtado, Maica; Pemberton, Alan; Wishart, Thomas M

    2015-11-01

    Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell

  12. The goat (Capra hircus) mammary gland secretory tissue proteome as influenced by weight loss: A study using label free proteomics

    DEFF Research Database (Denmark)

    Hernandez Castellano, Lorenzo E; Ferreira, Ana M.; Nanni, Paolo

    2016-01-01

    Seasonal weight loss (SWL) is a significant limitation to animal production. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Herein, label free proteomics was used to characterize the effects of SWL in two goat breeds with different levels of adaptatio...

  13. The goat (Capra hircus) mammary gland secretory tissue proteome as influenced by weight loss: A study using label free proteomics

    Science.gov (United States)

    Seasonal weight loss (SWL) is a significant limitation to animal production. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Herein, labelfree proteomics was used to characterize the effects of SWL in two goat breeds with different levels of adaptation to...

  14. Mass-Spectrometry-Based Proteomics Reveals Organ-Specific Expression Patterns To Be Used as Forensic Evidence.

    Science.gov (United States)

    Dammeier, Sascha; Nahnsen, Sven; Veit, Johannes; Wehner, Frank; Ueffing, Marius; Kohlbacher, Oliver

    2016-01-04

    Standard forensic procedures to examine bullets after an exchange of fire include a mechanical or ballistic reconstruction of the event. While this is routine to identify which projectile hit a subject by DNA analysis of biological material on the surface of the projectile, it is rather difficult to determine which projectile caused the lethal injury--often the crucial point with regard to legal proceedings. With respect to fundamental law it is the duty of the public authority to make every endeavor to solve every homicide case. To improve forensic examinations, we present a forensic proteomic method to investigate biological material from a projectile's surface and determine the tissues traversed by it. To obtain a range of relevant samples, different major bovine organs were penetrated with projectiles experimentally. After tryptic "on-surface" digestion, mass-spectrometry-based proteome analysis, and statistical data analysis, we were able to achieve a cross-validated organ classification accuracy of >99%. Different types of anticipated external variables exhibited no prominent influence on the findings. In addition, shooting experiments were performed to validate the results. Finally, we show that these concepts could be applied to a real case of murder to substantially improve the forensic reconstruction.

  15. SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1.

    Science.gov (United States)

    Yi, Zhou; Manil-Ségalen, Marion; Sago, Laila; Glatigny, Annie; Redeker, Virginie; Legouis, Renaud; Mucchielli-Giorgi, Marie-Hélène

    2016-05-06

    Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.

  16. Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer.

    Science.gov (United States)

    Delcourt, Vivian; Franck, Julien; Leblanc, Eric; Narducci, Fabrice; Robin, Yves-Marie; Gimeno, Jean-Pascal; Quanico, Jusal; Wisztorski, Maxence; Kobeissy, Firas; Jacques, Jean-François; Roucou, Xavier; Salzet, Michel; Fournier, Isabelle

    2017-07-01

    Recently, it was demonstrated that proteins can be translated from alternative open reading frames (altORFs), increasing the size of the actual proteome. Top-down mass spectrometry-based proteomics allows the identification of intact proteins containing post-translational modifications (PTMs) as well as truncated forms translated from reference ORFs or altORFs. Top-down tissue microproteomics was applied on benign, tumor and necrotic-fibrotic regions of serous ovarian cancer biopsies, identifying proteins exhibiting region-specific cellular localization and PTMs. The regions of interest (ROIs) were determined by MALDI mass spectrometry imaging and spatial segmentation. Analysis with a customized protein sequence database containing reference and alternative proteins (altprots) identified 15 altprots, including alternative G protein nucleolar 1 (AltGNL1) found in the tumor, and translated from an altORF nested within the GNL1 canonical coding sequence. Co-expression of GNL1 and altGNL1 was validated by transfection in HEK293 and HeLa cells with an expression plasmid containing a GNL1-FLAG (V5) construct. Western blot and immunofluorescence experiments confirmed constitutive co-expression of altGNL1-V5 with GNL1-FLAG. Taken together, our approach provides means to evaluate protein changes in the case of serous ovarian cancer, allowing the detection of potential markers that have never been considered. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  17. Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

    Science.gov (United States)

    Sandh, Gustaf; Ramström, Margareta; Stensjö, Karin

    2014-12-04

    In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments

  18. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

    Directory of Open Access Journals (Sweden)

    Schriemer David C

    2010-11-01

    Full Text Available Abstract Background Papaver somniferum (opium poppy is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a

  19. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

    Science.gov (United States)

    Desgagné-Penix, Isabel; Khan, Morgan F; Schriemer, David C; Cram, Dustin; Nowak, Jacek; Facchini, Peter J

    2010-11-18

    Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates

  20. Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb.

    Science.gov (United States)

    Domżalska, Lucyna; Kędracka-Krok, Sylwia; Jankowska, Urszula; Grzyb, Małgorzata; Sobczak, Mirosław; Rybczyński, Jan J; Mikuła, Anna

    2017-05-01

    Using cyto-morphological analysis of somatic embryogenesis (SE) in the tree fern Cyathea delgadii as a guide, we performed a comparative proteomic analysis in stipe explants undergoing direct SE. Plant material was cultured on hormone-free medium supplemented with 2% sucrose. Phenol extracted proteins were separated using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed for protein identification. A total number of 114 differentially regulated proteins was identified during early SE, i.e. when the first cell divisions started and several-cell pro-embryos were formed. Proteins were assigned to seven functional categories: carbohydrate metabolism, protein metabolism, cell organization, defense and stress responses, amino acid metabolism, purine metabolism, and fatty acid metabolism. Carbohydrate and protein metabolism were found to be the most sensitive SE functions with the greatest number of alterations in the intensity of spots in gel. Differences, especially in non-enzymatic and structural protein abundance, are indicative for cell organization, including cytoskeleton rearrangement and changes in cell wall components. The highest induced changes concern those enzymes related to fatty acid metabolism. Global analysis of the proteome reveals several proteins that can represent markers for the first 16days of SE induction and expression in fern. The findings of this research improve the understanding of molecular processes involved in direct SE in C. delgadii. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Proteomics of Fusarium oxysporum race 1 and race 4 reveals enzymes involved in carbohydrate metabolism and ion transport that might play important roles in banana Fusarium wilt.

    Science.gov (United States)

    Sun, Yong; Yi, Xiaoping; Peng, Ming; Zeng, Huicai; Wang, Dan; Li, Bo; Tong, Zheng; Chang, Lili; Jin, Xiang; Wang, Xuchu

    2014-01-01

    Banana Fusarium wilt is a soil-spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt.

  2. Interspecific proteomic comparisons reveal ash phloem genes potentially involved in constitutive resistance to the emerald ash borer.

    Science.gov (United States)

    Whitehill, Justin G A; Popova-Butler, Alexandra; Green-Church, Kari B; Koch, Jennifer L; Herms, Daniel A; Bonello, Pierluigi

    2011-01-01

    The emerald ash borer (Agrilus planipennis) is an invasive wood-boring beetle that has killed millions of ash trees since its accidental introduction to North America. All North American ash species (Fraxinus spp.) that emerald ash borer has encountered so far are susceptible, while an Asian species, Manchurian ash (F. mandshurica), which shares an evolutionary history with emerald ash borer, is resistant. Phylogenetic evidence places North American black ash (F. nigra) and Manchurian ash in the same clade and section, yet black ash is highly susceptible to the emerald ash borer. This contrast provides an opportunity to compare the genetic traits of the two species and identify those with a potential role in defense/resistance. We used Difference Gel Electrophoresis (DIGE) to compare the phloem proteomes of resistant Manchurian to susceptible black, green, and white ash. Differentially expressed proteins associated with the resistant Manchurian ash when compared to the susceptible ash species were identified using nano-LC-MS/MS and putative identities assigned. Proteomic differences were strongly associated with the phylogenetic relationships among the four species. Proteins identified in Manchurian ash potentially associated with its resistance to emerald ash borer include a PR-10 protein, an aspartic protease, a phenylcoumaran benzylic ether reductase (PCBER), and a thylakoid-bound ascorbate peroxidase. Discovery of resistance-related proteins in Asian species will inform approaches in which resistance genes can be introgressed into North American ash species. The generation of resistant North American ash genotypes can be used in forest ecosystem restoration and urban plantings following the wake of the emerald ash borer invasion.

  3. Proteomics as a tool for studying bacterial virulence and antimicrobial resistance

    Directory of Open Access Journals (Sweden)

    Francisco José Pérez -Llarena

    2016-03-01

    Full Text Available Proteomic studies have improved our understanding of the microbial world. The most recent advances in this field have helped us to explore aspects beyond genomics. For example, by studying proteins and their regulation, researchers now understand how some pathogenic bacteria have adapted to the lethal actions of antibiotics. Proteomics has also advanced our knowledge of mechanisms of bacterial virulence and some important aspects of how bacteria interact with human cells and, thus, of the pathogenesis of infectious diseases. This review article addresses these issues in some of the most important human pathogens. It also reports some applications of MALDI-TOF mass spectrometry that may be important for the diagnosis of bacterial resistance in clinical laboratories in the future. The reported advances will enable new diagnostic and therapeutic strategies to be developed in the fight against some of the most lethal bacteria affecting humans.

  4. Combined lipidomic and proteomic analysis of isolated human islets exposed to palmitate reveals time-dependent changes in insulin secretion and lipid metabolism.

    Directory of Open Access Journals (Sweden)

    Kirsten Roomp

    Full Text Available Studies on the pathophysiology of type 2 diabetes mellitus (T2DM have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our

  5. A novel approach to the study of the functional proteome in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Hennessy, Bryan; Lu, Yiling; Gonzalez-Angulo, Ana Maria; Carey, Mark; Myhre, Simen; Ju, Zhenlin; Coombes, Kevin; Meric-Bernstam, Funda; Bedrosian, Isabelle; Davies, Michael A.; Siwak, Doris; Agarwal, Roshan; Zhang, Fan; Overgaard, Jens; Alsner, Jan; Neve, Richard M.; Kuo, Wen-Lin; Gray, Joe W.; Borresen-Dale, Anne-Lise; Mills, Gordon B.

    2008-10-10

    Factors including intratumoral heterogeneity and variability in tissue handling potentially hamper the application of reverse phase protein arrays (RPPA) to study of the solid tumor functional proteome. To address this, RPPA was applied to quantify protein expression and activation in 233 human breast tumors and 52 breast cancer cell lines. Eighty-two antibodies that recognize kinase and steroid signaling events and their effectors were validated for RPPA because of the importance of these proteins to breast carcinogenesis. Reproducibility in replicate lysates was excellent. Intratumoral protein expression was less variable than intertumoral expression, and prognostic biomarkers retained the ability to accurately predict patient outcomes when analyzed in different tumor sites. Although 21/82 total and phosphoproteins demonstrated time-dependent instability in breast tumors that were placed at room temperature after surgical excision for 24 hours prior to freezing, the functional proteomic 'fingerprint' was robust in most tumors until at least 24 hours before tissue freezing. Correlations between RPPA and immunohistochemistry were statistically significant for assessed proteins but RPPA demonstrated a superior dynamic range and detected, for example, an 866-fold difference in estrogen receptor alpha level across breast tumors. Protein and mRNA levels were concordant (at p {le} 0.05) for 41.3% and 61.1% of assayed targets in breast tumors and cell lines, respectively. Several phosphorylation and cleavage products did not correlate with the corresponding transcript levels. In conclusion, the reproducibility of RPPA, the faithfulness with which proteins and the functional proteomic 'fingerprint' are preserved in different sections derived from primary breast tumors, and the surprising stability of this 'fingerprint' with increasing time to freezing all facilitate the application of RPPA to the accurate study of protein biomarkers in non

  6. Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

    DEFF Research Database (Denmark)

    Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.

    2014-01-01

    Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes...... involved in the Mendelian disorder long QT syndrome (LOTS). We integrated the LOTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LOTS protein...... network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy...

  7. A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance.

    Science.gov (United States)

    Wu, Chongde; Zhang, Juan; Chen, Wei; Wang, Miao; Du, Guocheng; Chen, Jian

    2012-01-01

    Lactobacillus casei has traditionally been recognized as a probiotic and frequently used as an adjunct culture in fermented dairy products, where acid stress is an environmental condition commonly encountered. In the present study, we carried out a comparative physiological and proteomic study to investigate lactic-acid-induced alterations in Lactobacillus casei Zhang (WT) and its acid-resistant mutant. Analysis of the physiological data showed that the mutant exhibited 33.8% higher glucose phosphoenolpyruvate:sugar phosphotransferase system activity and lower glycolytic pH compared with the WT under acidic conditions. In addition, significant differences were detected in both cells during acid stress between intracellular physiological state, including intracellular pH, H(+)-ATPase activity, and intracellular ATP pool. Comparison of the proteomic data based on 2D-DIGE and i-TRAQ indicated that acid stress invoked a global change in both strains. The mutant protected the cells against acid damage by regulating the expression of key proteins involved in cellular metabolism, DNA replication, RNA synthesis, translation, and some chaperones. Proteome results were validated by Lactobacillus casei displaying higher intracellular aspartate and arginine levels, and the survival at pH 3.3 was improved 1.36- and 2.10-fold by the addition of 50-mM aspartate and arginine, respectively. To our knowledge, this is the first demonstration that aspartate may be involved in acid tolerance in Lactobacillus casei. Results presented here may help us understand acid resistance mechanisms and help formulate new strategies to enhance the industrial applications of this species.

  8. Understanding mechanism of sea cucumber Apostichopus japonicus aestivation: Insights from TMT-based proteomic study.

    Science.gov (United States)

    Chen, Muyan; Li, Xingke; Zhu, Aijun; Storey, Kenneth B; Sun, Lina; Gao, Tianxiang; Wang, Tianming

    2016-09-01

    Marine invertebrate aestivation is a unique strategy for summer survival in response to hot marine conditions. The sea cucumber, Apostichopus japonicus, is an excellent model marine invertebrate for studies of environmentally-induced aestivation. In the present study, we used a tandem mass tag (TMT)-coupled LC-MS/MS approach to identify and quantify the global proteome expression profile over the aestivation-arousal cycle of A. japonicus. A total of 3920 proteins were identified from the intestine of sea cucumber. Among them, 630 proteins showed significant differential expression when comparing three conditions of sea cucumbers: non-aestivating (active), deep-aestivation (at least 15days of continuous aestivation), and arousal after aestivation (renewed moving and feeding). Sea cucumbers in deep aestivation showed substantial differentially expressed proteins (143 up-regulated and 267 down-regulated proteins compared with non-aestivating controls). These differentially expressed proteins suggested that protein and phospholipid probably are major fuel sources during hypometabolism and a general attenuation of carbohydrate metabolism was observed during deep aestivation. Differentially expressed proteins also provided the first global picture of a shift in protein synthesis, protein folding, DNA binding, apoptosis, cellular transport and signaling, and cytoskeletal proteins during deep aestivation in sea cucumbers. A comparison of arousal from aestivation with deep aestivation, revealed a general reversal of the changes that occurred in aestivation for most proteins. Western blot detection further validated the significant up-regulation of HSP70 and down-regulation of methyltransferase-like protein 7A-like in deep-aestivation. Our results suggest that there is substantial post-transcriptional regulation of proteins during the aestivation-arousal cycle in sea cucumbers. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Studying Different Clinical Syndromes Of Paediatric Severe Malaria Using Plasma Proteomics

    KAUST Repository

    Ramaprasad, Abhinay

    2012-08-01

    Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

  10. The regulation mechanisms of soluble starch and glycerol for production of azaphilone pigments in Monascus purpureus FAFU618 as revealed by comparative proteomic and transcriptional analyses.

    Science.gov (United States)

    Huang, Zi-Rui; Zhou, Wen-Bin; Yang, Xue-Ling; Tong, Ai-Jun; Hong, Jia-Li; Guo, Wei-Ling; Li, Tian-Tian; Jia, Rui-Bo; Pan, Yu-Yang; Lin, Jun; Lv, Xu-Cong; Liu, Bin

    2018-04-01

    Monascus spp. have been used for thousands of years as a traditional food additive in China. This mold can produce many different types of commercially valuable secondary metabolites of biological activity. Soluble starch and glycerol are the two principal carbon sources universally utilized by Monascus for the production of beneficial metabolites. In this study, the effects and regulation mechanisms of soluble starch and glycerol for M. purpureus FAFU618 on Monascus azaphilone pigments (MonAzPs) were investigated through ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS), comparative proteomics and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). The production of intracellular and extracellular pigments was significantly different between the soluble starch group (SSG) and glycerol group (GCG). Additionally, the components of intracellular pigments revealed by UPLC-QTOF-MS/MS showed that Monascin and Ankaflavin increased significantly in the GCG, while Rubropunctatin and Monascorubrin increased in the SSG. Differentially expressed proteins of mycelia between SSG and GCG were analyzed by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS. We identified 27 proteins with statistically altered expression, of which 18 proteins associated with the EMP (glycolytic pathway), translation, energy generation, proteolysis, etc. were up-regulated, and 9 proteins, including ribosomal proteins, heat shock proteins (HSPs) and others, were down-regulated in GCG. Meanwhile, the expression levels of MonAzP biosynthetic genes were also analyzed by RT-qPCR, and the results showed that mppA, mppC, mppR1 and mppR2 were down-regulated, whereas genes MpPKS5, MpFasA2, MpFasB2, mppB, mppD and mppE were up-regulated. Collectively, these findings illustrate that the regulation of MonAzPs is not only closely related to the expression levels of certain proteins in the polyketide synthesis pathway

  11. Proteomic investigation of Vibrio alginolyticus challenged Caenorhabditis elegans revealed regulation of cellular homeostasis proteins and their role in supporting innate immune system.

    Science.gov (United States)

    Durai, Sellegounder; Singh, Nirpendra; Kundu, Suman; Balamurugan, Krishnaswamy

    2014-08-01

    Caenorhabditis elegans has been the preferred model system for many investigators to study pathogenesis. In the present investigation, regulation of C. elegans proteome was explored against V. alginolyticus infection using quantitative proteomics approach. Proteins were separated using 2D-DIGE and the differentially regulated proteins were identified using PMF and MALDI TOF/TOF analysis. The results thus obtained were validated using Western blotting for candidate proteins. The corresponding transcriptional regulation was quantified subsequently using real-time PCR. Interaction network for candidate proteins was predicted using search tool for the retrieval of interacting genes/proteins (STRING) and functional validation was performed using respective mutant strains. Out of the 25 proteins identified, 21 proteins appeared to be upregulated while four were downregulated. Upregulated proteins included those involved in stress-response (PDI-2, HSP-6), immune-response (protein kinase -18, GST-8) and energy-production (ATP-2) while proteins involved in structural maintenance (IFB-2) and lipid metabolism (SODH-1) were downregulated. The roles of these players in the host system during Vibrio infection was analyzed in vivo using wild type and mutant C. elegans. Survival assays using mutants lacking pdi-2, ire-1, and xbp-1 displayed enhanced susceptibility to V. alginolyticus. Cellular stress generated by V. alginolyticus was determined using ROS assay. This is the first report of proteome changes in C. elegans against V. alginolyticus challenge and highlights the significance of unfolded protein response (UPR) pathway during bacterial infection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling.

    Science.gov (United States)

    Chowdhury, Saiful M; Zhu, Xuewei; Aloor, Jim J; Azzam, Kathleen M; Gabor, Kristin A; Ge, William; Addo, Kezia A; Tomer, Kenneth B; Parks, John S; Fessler, Michael B

    2015-07-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1(-/-) macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1(+/+) and Abca1(-/-) macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1(+/+) macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1(-/-) rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. © 2015 by The American Society for Biochemistry and

  13. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

    Science.gov (United States)

    Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

    2015-01-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

  14. Proteomic approach in human health and disease: Preventive and cure studies

    Directory of Open Access Journals (Sweden)

    Khaled MM Koriem

    2018-01-01

    Full Text Available Proteomic is a branch of science that deals with various numbers of proteins where proteins are essential human constituents. Proteomic has a lot of functions inside the human and animal living organisms. This review helps to make a thought on the importance of proteomic application in human health and disease with special reference to preventive and cure studies. The human health can be divided into physical and mental health. The physical health relates to keeping human body state in a good health and to nutritional type and environmental factors. The mental health correlates to human psychological state. The main factors that affect the status of human health are human diet, exercise and sleep. The healthy diet is very important and needs to maintain the human health. The training program exercise improves human fitness and overall health and wellness. The sleep is a vital factor to sustain the human health. The human disease indicates abnormal human condition which influences the specific human part or the whole human body. There are external and internal factors which induce human disease. The external factors include pathogens while internal factors include allergies and autoimmunity. There are 4 principle types of human diseases: (1 infectious disease, (2 deficiency disease, (3 genetic disease and (4 physiological disease. There are many and various external microbes' factors that induce human infectious disease and these agents include viruses, bacteria, fungi and protozoa. The lack of necessary and vital dietary rudiments such as vitamins and minerals is the main cause of human deficiency disease. The genetic disease is initiated by hereditary disturbances that occur in the human genetic map. The physiological disease occurs when the normal human function body is affected due to human organs become malfunction. In conclusion, proteomic plays a vital and significant role in human health and disease.

  15. Proteomic-Based Approaches for the Study of Cytokines in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ángela Marrugal

    2016-01-01

    Full Text Available Proteomic techniques are currently used to understand the biology of different human diseases, including studies of the cell signaling pathways implicated in cancer progression, which is important in knowing the roles of different proteins in tumor development. Due to its poor prognosis, proteomic approaches are focused on the identification of new biomarkers for the early diagnosis, prognosis, and targeted treatment of lung cancer. Cytokines are proteins involved in inflammatory processes and have been proposed as lung cancer biomarkers and therapeutic targets because it has been reported that some cytokines play important roles in tumor development, invasion, and metastasis. In this review, we aim to summarize the different proteomic techniques used to discover new lung cancer biomarkers and therapeutic targets. Several cytokines have been identified as important players in lung cancer using these techniques. We underline the most important cytokines that are useful as biomarkers and therapeutic targets. We also summarize some of the therapeutic strategies targeted for these cytokines in lung cancer.

  16. Mass Spectrometric Analyses Reveal a Central Role for Ubiquitylation in Remodeling the Arabidopsis Proteome during Photomorphogenesis.

    Science.gov (United States)

    Aguilar-Hernández, Victor; Kim, Do-Young; Stankey, Robert J; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

    2017-06-05

    The switch from skotomorphogenesis to photomorphogenesis is a key developmental transition in the life of seed plants. While much of the underpinning proteome remodeling is driven by light-induced changes in gene expression, the proteolytic removal of specific proteins by the ubiquitin-26S proteasome system is also likely paramount. Through mass spectrometric analysis of ubiquitylated proteins affinity-purified from etiolated Arabidopsis seedlings before and after red-light irradiation, we identified a number of influential proteins whose ubiquitylation status is modified during this switch. We observed a substantial enrichment for proteins involved in auxin, abscisic acid, ethylene, and brassinosteroid signaling, peroxisome function, disease resistance, protein phosphorylation and light perception, including the phytochrome (Phy) A and phototropin photoreceptors. Soon after red-light treatment, PhyA becomes the dominant ubiquitylated species, with ubiquitin attachment sites mapped to six lysines. A PhyA mutant protected from ubiquitin addition at these sites is substantially more stable in planta upon photoconversion to Pfr and is hyperactive in driving photomorphogenesis. However, light still stimulates ubiquitylation and degradation of this mutant, implying that other attachment sites and/or proteolytic pathways exist. Collectively, we expand the catalog of ubiquitylation targets in Arabidopsis and show that this post-translational modification is central to the rewiring of plants for photoautotrophic growth. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  17. Comparative proteomic analysis reveals molecular mechanism of seedling roots of different salt tolerant soybean genotypes in responses to salinity stress

    Directory of Open Access Journals (Sweden)

    Hongyu Ma

    2014-09-01

    Full Text Available Salinity stress is one of the major abiotic stresses that limit agricultural yield. To understand salt-responsive protein networks in soybean seedling, the extracted proteins from seedling roots of two different genotypes (Lee 68 and Jackson were analyzed under salt stress by two-dimensional polyacrylamide gel electrophoresis. Sixty-eight differentially expressed proteins were detected and identified. The identified proteins were involved in 13 metabolic pathways and cellular processes. Proteins correlated to brassinosteroid and gilbberellin signalings were significantly increased only in the genotype Lee 68 under salt stress; abscisic acid content was positively correlated with this genotype; proteins that can be correlated to Ca2+ signaling were more strongly enhanced by salt stress in the seedling roots of genotype Lee 68 than in those of genotype Jackson; moreover, genotype Lee 68 had stronger capability of reactive oxygen species scavenging and cell K+/Na+ homeostasis maintaining in seedling roots than genotype Jackson under salt stress. Since the genotype Lee 68 has been described in literature as being tolerant and Jackson as sensitive, we hypothesize that these major differences in the genotype Lee 68 might contribute to salt tolerance. Combined with our previous comparative proteomics analysis on seedling leaves, the similarities and differences between the salt-responsive protein networks found in the seedling leaves and roots of both the genotypes were discussed. Such a result will be helpful in breeding of salt-tolerant soybean cultivars.

  18. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    KAUST Repository

    An, Xiaomeng

    2017-06-21

    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

  19. Comparative Physiological and Proteomic Analysis Reveals the Leaf Response to Cadmium-Induced Stress in Poplar (Populus yunnanensis.

    Directory of Open Access Journals (Sweden)

    Yunqiang Yang

    Full Text Available Excess amounts of heavy metals are important environmental pollutants with significant ecological and nutritional effects. Cdmium (Cd is of particular concern because of its widespread occurrence and high toxicity. We conducted physiological and proteomic analyses to improve our understanding of the responses of Populus yunnanensis to Cd stress. The plantlets experienced two apparent stages in their response to Cd stress. During the first stage, transiently induced defense-response molecules, photosynthesis- and energy-associated proteins, antioxidant enzymes and heat shock proteins (HSPs accumulated to enhance protein stability and establish a new cellular homeostasis. This activity explains why plant photosynthetic capability during this period barely changed. During the second stage, a decline of ribulose-1, 5-bisphosphate carboxylase (RuBisCO and HSP levels led to imbalance of the plant photosynthetic system. Additionally, the expression of Mitogen-activated protein kinase 3 (MPK3, Mitogen-activated protein kinase 6 (MPK6 and a homeobox-leucine zipper protein was higher in the second stage. Higher expression of caffeoyl-CoA O-methyltransferase (CCoAOMT may regulate plant cell wall synthesis for greater Cd storage. These genes may be candidates for further research and use in genetic manipulation of poplar tolerance to Cd stress.

  20. Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

    Science.gov (United States)

    2016-01-01

    Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions. PMID:27654267

  1. Toxic effects of male Perna viridis gonad exposed to BaP, DDT and their mixture: A metabolomic and proteomic study of the underlying mechanism.

    Science.gov (United States)

    Song, Qinqin; Zheng, Pengfei; Qiu, Liguo; Jiang, Xiu; Zhao, Hongwei; Zhou, Hailong; Han, Qian; Diao, Xiaoping

    2016-01-05

    Benzo(a)pyrene and dichlorodiphenyltrichloroethane are typical persistent organic pollutants, and also the widespread environmental estrogens with known toxicity towards green mussels Perna viridis. In this study, the toxicological effects of BaP and DDT and their mixture were assessed in green mussel gonads using proteomic and metabolomic approaches. Metabolomics by NMR spectroscopy revealed that BaP did not show obvious metabolite changes in the gonad of male green mussel. DDT mainly caused some disturbance of osmotic regulation and energy metabolism by changing BCAAs, alanine, threonine, arginine, etc., unknown metabolite (3.53 ppm), glycine, homarine and ATP at different levels. However, the mixture of BaP and DDT mainly caused some disturbance in osmotic regulation and energy metabolism by differentially altering branched chain amino acids, glutamate, alanine, arginine, unknown metabolite (3.53 ppm), glycine, 4-aminobutyrate, dimethylglycine, homarine and ATP. The results suggest that DDT alone may cause most of metabolites changes in the mixture exposed male mussel gonad, and the results also show that the male P. viridis gonad was more sensitive to DDT than BaP exposures. Proteomic study showed that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced signal transduction, oxidative stress, spermatogenesis, etc. in the male green mussel gonad; whereas DDT exposure altered proteins that were associated with signal transduction, oxidative stress, cytoskeleton and cell structure, cellular organization, energy metabolism, etc. However, the mixture of BaP and DDT affected proteins related to cytoskeleton and cell structure, oxidative stress, cellular organization, etc. This research demonstrated that metabolomic and proteomic approaches could better elucidate the underlying mechanism of environmental pollutants gonad toxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Comparative physiological and proteomic analyses reveal the actions of melatonin in the reduction of oxidative stress in Bermuda grass (Cynodon dactylon (L). Pers.).

    Science.gov (United States)

    Shi, Haitao; Wang, Xin; Tan, Dun-Xian; Reiter, Russel J; Chan, Zhulong

    2015-08-01

    The fact of melatonin as an important antioxidant in animals led plant researchers to speculate that melatonin also acts in the similar manner in plants. Although melatonin has significant effects on alleviating stress-triggered reactive oxygen species (ROS), the involvement of melatonin in direct oxidative stress and the underlying physiological and molecular mechanisms remain unclear in plants. In this study, we found that exogenous melatonin significantly alleviated hydrogen peroxide (H2O2)-modulated plant growth, cell damage, and ROS accumulation in Bermuda grass. Additionally, 76 proteins significantly influenced by melatonin during mock or H2O2 treatment were identified by gel-free proteomics using iTRAQ (isobaric tags for relative and absolute quantitation). Metabolic pathway analysis showed that several pathways were markedly enhanced by melatonin and H2O2 treatments, including polyamine metabolism, ribosome pathway, major carbohydrate metabolism, photosynthesis, redox, and amino acid metabolism. Taken together, this study provides more comprehensive insights into the physiological and molecular mechanisms of melatonin in Bermuda grass responses to direct oxidative stress. This may relate to the activation of antioxidants, modulation of metabolic pathways, and extensive proteome reprograming. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sylvie Cornelie

    Full Text Available Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

  4. Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

    Science.gov (United States)

    Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

    2015-07-01

    The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Plasma membrane proteomic analysis of human Gastric Cancer tissues: revealing flotillin 1 as a marker for Gastric Cancer

    International Nuclear Information System (INIS)

    Gao, Wen; Xu, Jing; Wang, Fuqiang; Zhang, Long; Peng, Rui; Shu, Yongqian; Wu, Jindao; Tang, Qiyun; Zhu, Yunxia

    2015-01-01

    Gastric cancer remains the second leading cause of cancer-related deaths in the world. Successful early gastric cancer detection is hampered by lack of highly sensitive and specific biomarkers. Plasma membrane proteins participate and/or have a central role in the metastatic process of cancer cells and are potentially useful for cancer therapy due to easy accessibility of the targets. In the present research, TMT method followed by mass spectrometry analysis was used to compare the relative expression levels of plasma membrane proteins between noncancer and gastric cancer tissues. Of a total data set that included 501 identified proteins, about 35% of the identified proteins were found to be plasma membrane and associated proteins. Among them, 82 proteins were at least 1.5-fold up- or down-regulated in gastric cancer compared with the adherent normal tissues. A number of markers (e.g. annexin A6, caveolin 1, epidermal growth factor receptor, integrin beta 4) were previously reported as biomarkers of GC. Additionally, several potential biomarkers participated in endocytosis pathway and integrin signaling pathways were firstly identified as differentially expressed proteins in GC samples. Our findings also supported the notion that flotillin 1 is a potential biomarker that could be exploited for molecular imaging-based detection of gastric cancer. Together, the results show that subcellular proteomics of tumor tissue is a feasible and promising avenue for exploring oncogenesis. The online version of this article (doi:10.1186/s12885-015-1343-5) contains supplementary material, which is available to authorized users

  6. Proteomics analysis of maize (Zea mays L.) grain based on iTRAQ reveals molecular mechanisms of poor grain filling in inferior grains.

    Science.gov (United States)

    Yu, Tao; Li, Geng; Liu, Peng; Dong, Shuting; Zhang, Jiwang; Zhao, Bin

    2017-06-01

    In maize, inferior grains (IG) located on the upper part of the ear have poor grain filling process compared to superior grains (SG) located on the middle and lower parts of the ear. This difference limits satisfactory yield and quality; however, the underlying molecular mechanisms remain unknown. Here, using the isobaric tag for relative and absolute quantification (iTRAQ) technology, the proteomes of IG and SG during early and middle grain filling stages were investigated. In total, 4720 proteins were identified in maize grain and 305 differentially accumulated proteins (DiAPs) were detected between IG and SG. These DiAPs were involved in diverse cellular and metabolic processes with preferred distribution in protein synthesis/destination and metabolism. Compared to SG, DiAPs related to cell growth/division and starch synthesis were lag-accumulated and down-regulated in IG, respectively, resulting in smaller sink sizes and lower sink activities in IG. Meanwhile, impediment of the glycolysis pathway in IG may lead to reduce energy supply and building materials for substance synthesis. Additionally, reactive oxygen species (ROS) homeostasis and the defense system were disturbed in IG, which might lead to reduce protection against various environmental stresses. The present study provides new information on the proteomic differences between IG and SG, and explains possible molecular mechanisms for poor grain filling in IG. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.; Smolders, Arne; Brewer, Heather M.; Camp, David G.; Smith, Richard D.; Braeckman, Bart P.

    2014-04-04

    The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. Finally, this restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.

  8. LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.; Smolders, Arne; Brewer, Heather M.; Camp, David G.; Smith, Richard D.; Braeckman, Bart P.

    2014-02-20

    The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediary metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.

  9. Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren's syndrome-associated dry eye.

    Science.gov (United States)

    Zhou, Lei; Wei, Ruihua; Zhao, Ping; Koh, Siew Kwan; Beuerman, Roger W; Ding, Chuanqing

    2013-08-01

    Sjögren's syndrome (SS) is an autoimmune disease that results in pathological dryness of mouth and eye. The diagnosis of SS depends on both clinical evaluation and specific antibodies. The goal of this study was to use quantitative proteomics to investigate changes in tear proteins in a rabbit model of SS-associated dry eye, induced autoimmune dacryoadenitis (IAD). Proteomic analysis was performed by iTRAQ and nano LC-MS/MS on tears collected from the ocular surface, and specific proteins were verified by high resolution MRM. It was found that in the tears of IAD rabbits at 2 and 4 weeks after induction, S100 A6, S100 A9, and serum albumin were upregulated, whereas serotransferrin (TF), prolactin-inducible protein (PIP), polymeric immunoglobulin receptor (pIgR), and Ig gamma chain C region were downregulated. High resolution MRM with mTRAQ labeling verified the changes in S100 A6, TF, PIP, and pIgR. Our results indicated significant changes of tear proteins in IAD rabbits, suggesting these proteins could potentially be used as biomarkers for the diagnosis and prognosis of dry eye. Several of these proteins were also found in the tears of non-SS dry eye patients indicating a common basis of ocular surface pathology, however, pIgR appears to be unique to SS. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, during short-term exposure to irradiance stress reveals significant down regulation of several heat-shock proteins.

    Science.gov (United States)

    Mahong, Bancha; Roytrakul, Suttiruk; Phaonaklop, Narumon; Wongratana, Janewit; Yokthongwattana, Kittisak

    2012-03-01

    Oxygenic photosynthetic organisms often suffer from excessive irradiance, which cause harmful effects to the chloroplast proteins and lipids. Photoprotection and the photosystem II repair processes are the mechanisms that plants deploy to counteract the drastic effects from irradiance stress. Although the protective and repair mechanisms seemed to be similar in most plants, many species do confer different level of tolerance toward high light. Such diversity may originate from differences at the molecular level, i.e., perception of the light stress, signal transduction and expression of stress responsive genes. Comprehensive analysis of overall changes in the total pool of proteins in an organism can be performed using a proteomic approach. In this study, we employed 2-DE/LC-MS/MS-based comparative proteomic approach to analyze total proteins of the light sensitive model unicellular green alga Chlamydomonas reinhardtii in response to excessive irradiance. Results showed that among all the differentially expressed proteins, several heat-shock proteins and molecular chaperones were surprisingly down-regulated after 3-6 h of high light exposure. Discussions were made on the possible involvement of such down regulation and the light sensitive nature of this model alga.

  11. Comparative proteomic and physiological analyses reveal the protective effect of exogenous polyamines in the bermudagrass (Cynodon dactylon) response to salt and drought stresses.

    Science.gov (United States)

    Shi, Haitao; Ye, Tiantian; Chan, Zhulong

    2013-11-01

    Polyamines conferred enhanced abiotic stress tolerance in multiple plant species. However, the effect of polyamines on abiotic stress and physiological change in bermudagrass, the most widely used warm-season turfgrasses, are unknown. In this study, pretreatment of exogenous polyamine conferred increased salt and drought tolerances in bermudagrass. Comparative proteomic analysis was performed to further investigate polyamines mediated responses, and 36 commonly regulated proteins by at least two types of polyamines in bermudagrass were successfully identified, including 12 proteins with increased level, 20 proteins with decreased level and other 4 specifically expressed proteins. Among them, proteins involved in electron transport and energy pathways were largely enriched, and nucleoside diphosphate kinase (NDPK) and three antioxidant enzymes were extensively regulated by polyamines. Dissection of reactive oxygen species (ROS) levels indicated that polyamine-derived H2O2 production might play dual roles under abiotic stress conditions. Moreover, accumulation of osmolytes was also observed after application of exogenous polyamines, which is consistent with proteomics results that several proteins involved in carbon fixation pathway were mediated commonly by polyamines pretreatment. Taken together, we proposed that polyamines could activate multiple pathways that enhance bermudagrass adaption to salt and drought stresses. These findings might be applicable for genetically engineering of grasses and crops to improve stress tolerance.

  12. LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

    Science.gov (United States)

    2015-01-01

    The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity. PMID:24555535

  13. Global Proteomics Revealed Klebsiella pneumoniae Induced Autophagy and Oxidative Stress in Caenorhabditis elegans by Inhibiting PI3K/AKT/mTOR Pathway during Infection

    Directory of Open Access Journals (Sweden)

    Arumugam Kamaladevi

    2017-09-01

    Full Text Available The enterobacterium, Klebsiella pneumoniae invades the intestinal epithelium of humans by interfering with multiple host cell response. To uncover a system-level overview of host response during infection, we analyzed the global dynamics of protein profiling in Caenorhabditis elegans using quantitative proteomics approach. Comparison of protein samples of nematodes exposed to K. pneumoniae for 12, 24, and 36 h by 2DE revealed several changes in host proteome. A total of 266 host-encoded proteins were identified by 2DE MALDI-MS/MS and LC-MS/MS and the interacting partners of the identified proteins were predicted by STRING 10.0 analysis. In order to understand the interacting partners of regulatory proteins with similar or close pI ranges, a liquid IEF was performed and the isolated fractions containing proteins were identified by LC-MS/MS. Functional bioinformatics analysis on identified proteins deciphered that they were mostly related to the metabolism, dauer formation, apoptosis, endocytosis, signal transduction, translation, developmental, and reproduction process. Gene enrichment analysis suggested that the metabolic process as the most overrepresented pathway regulated against K. pneumoniae infection. The dauer-like formation in infected C. elegans along with intestinal atrophy and ROS during the physiological analysis indicated that the regulation of metabolic pathway is probably through the involvement of mTOR. Immunoblot analysis supported the above notion that the K. pneumoniae infection induced protein mis-folding in host by involving PI3Kinase/AKT-1/mTOR mediated pathway. Furthermore, the susceptibility of pdi-2, akt-1, and mTOR C. elegans mutants confirmed the role and involvement of PI3K/AKT/mTOR pathway in mediating protein mis-folding which appear to be translating the vulnerability of host defense toward K. pneumoniae infection.

  14. Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

    Science.gov (United States)

    Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Núñez, Félix; Asensio, Miguel A

    2017-09-01

    The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl 2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl 2 -incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl 2 . Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Proteomic analysis of Herbaspirillum seropedicae reveals ammonium-induced AmtB-dependent membrane sequestration of PII proteins.

    Science.gov (United States)

    Huergo, Luciano F; Noindorf, Lilian; Gimenes, Camila; Lemgruber, Renato S P; Cordellini, Daniela F; Falarz, Lucas J; Cruz, Leonardo M; Monteiro, Rose A; Pedrosa, Fábio O; Chubatsu, Leda S; Souza, Emanuel M; Steffens, Maria B R

    2010-07-01

    This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

  16. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-03-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

  17. Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

    Science.gov (United States)

    Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

    2016-01-01

    Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery. PMID:26956660

  18. Proteomic and transcriptomic studies of HBV-associated liver fibrosis of an AAV-HBV-infected mouse model.

    Science.gov (United States)

    Kan, Fangming; Ye, Lei; Yan, Tao; Cao, Jiaqi; Zheng, Jianhua; Li, Wuping

    2017-08-22

    Human hepatitis B virus (HBV) infection is an important public health issue in the Asia-Pacific region and is associated with chronic hepatitis, liver fibrosis, cirrhosis and even liver cancer. However, the underlying mechanisms of HBV-associated liver fibrosis remain incompletely understood. In the present study, proteomic and transcriptomic approaches as well as biological network analyses were performed to investigate the differentially expressed molecular signature and key regulatory networks that were associated with HBV-mediated liver fibrosis. RNA sequencing and 2DE-MALDI-TOF/TOF were performed on liver tissue samples obtained from HBV-infected C57BL/6 mouse generated via AAV8-HBV virus. The results showed that 322 genes and 173 proteins were differentially expressed, and 28 HBV-specific proteins were identified by comprehensive proteomic and transcriptomic analysis. GO analysis indicated that the differentially expressed proteins were predominantly involved in oxidative stress, which plays a key role in HBV-related liver fibrosis. Importantly, CAT, PRDX1, GSTP1, NXN and BLVRB were shown to be associated with oxidative stress among the differentially expressed proteins. The most striking results were validated by Western blot and RT-qPCR. The RIG-I like receptor signaling pathway was found to be the major signal pathway that changed during HBV-related fibrosis. This study provides novel insights into HBV-associated liver fibrosis and reveals the significant role of oxidative stress in liver fibrosis. Furthermore, CAT, BLVRB, NXN, PRDX1, and IDH1 may be candidates for detection of liver fibrosis or therapeutic targets for the treatment of liver fibrosis.

  19. PeptideManager: A Peptide Selection Tool for Targeted Proteomic Studies Involving Mixed Samples from Different Species

    Directory of Open Access Journals (Sweden)

    Kevin eDemeure

    2014-09-01

    Full Text Available The search for clinically useful protein biomarkers using advanced mass spectrometry approaches represents a major focus in cancer research. However, the direct analysis of human samples may be challenging due to limited availability, the absence of appropriate control samples, or the large background variability observed in patient material. As an alternative approach, human tumors orthotopically implanted into a different species (xenografts are clinically relevant models that have proven their utility in pre-clinical research. Patient derived xenografts for glioblastoma have been extensively characterized in our laboratory and have been shown to retain the characteristics of the parental tumor at the phenotypic and genetic level. Such models were also found to adequately mimic the behavior and treatment response of human tumors. The reproducibility of such xenograft models, the possibility to identify their host background and perform tumor-host interaction studies, are major advantages over the direct analysis of human samples.At the proteome level, the analysis of xenograft samples is challenged by the presence of proteins from two different species which, depending on tumor size, type or location, often appear at variable ratios. Any proteomics approach aimed at quantifying proteins within such samples must consider the identification of species specific peptides in order to avoid biases introduced by the host proteome. Here, we present an in-house methodology and tool developed to select peptides used as surrogates for protein candidates from a defined proteome (e.g., human in a host proteome background (e.g., mouse, rat suited for a mass spectrometry analysis. The tools presented here are applicable to any species specific proteome, provided a protein database is available. By linking the information from both proteomes, PeptideManager significantly facilitates and expedites the selection of peptides used as surrogates to analyze

  20. Expression proteomics study to determine metallodrug targets and optimal drug combinations.

    Science.gov (United States)

    Lee, Ronald F S; Chernobrovkin, Alexey; Rutishauser, Dorothea; Allardyce, Claire S; Hacker, David; Johnsson, Kai; Zubarev, Roman A; Dyson, Paul J

    2017-05-08

    The emerging technique termed functional identification of target by expression proteomics (FITExP) has been shown to identify the key protein targets of anti-cancer drugs. Here, we use this approach to elucidate the proteins involved in the mechanism of action of two ruthenium(II)-based anti-cancer compounds, RAPTA-T and RAPTA-EA in breast cancer cells, revealing significant differences in the proteins upregulated. RAPTA-T causes upregulation of multiple proteins suggesting a broad mechanism of action involving suppression of both metastasis and tumorigenicity. RAPTA-EA bearing a GST inhibiting ethacrynic acid moiety, causes upregulation of mainly oxidative stress related proteins. The approach used in this work could be applied to the prediction of effective drug combinations to test in cancer chemotherapy clinical trials.

  1. Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

    Science.gov (United States)

    Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

    2015-10-14

    than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

  2. Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

    International Nuclear Information System (INIS)

    Yu, Qing; Shi, Xudong; Feng, Yu; Kent, K. Craig; Li, Lingjun

    2017-01-01

    Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

  3. Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Qing [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Shi, Xudong [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Feng, Yu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Kent, K. Craig [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Li, Lingjun, E-mail: lingjun.li@wisc.edu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Department of Chemistry, University of Wisconsin, Madison, WI 53706 (United States)

    2017-05-22

    Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

  4. Proteomic analysis of membrane microdomain-associated proteins in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder reveals alterations in LAMP, STXBP1 and BASP1 protein expression.

    LENUS (Irish Health Repository)

    Behan, A T

    2009-06-01

    The dorsolateral prefrontal cortex (dlpfc) is strongly implicated in the pathogenesis of schizophrenia (SCZ) and bipolar disorder (BPD) and, within this region, abnormalities in glutamatergic neurotransmission and synaptic function have been described. Proteins associated with these functions are enriched in membrane microdomains (MM). In the current study, we used two complementary proteomic methods, two-dimensional difference gel electrophoresis and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by reverse phase-liquid chromatography-tandem mass spectrometry (RP-LC-MS\\/MS) (gel separation liquid chromatography-tandem mass spectrometry (GeLC-MS\\/MS)) to assess protein expression in MM in pooled samples of dlpfc from SCZ, BPD and control cases (n=10 per group) from the Stanley Foundation Brain series. We identified 16 proteins altered in one\\/both disorders using proteomic methods. We selected three proteins with roles in synaptic function (syntaxin-binding protein 1 (STXBP1), brain abundant membrane-attached signal protein 1 (BASP1) and limbic system-associated membrane protein (LAMP)) for validation by western blotting. This revealed significantly increased expression of these proteins in SCZ (STXBP1 (24% difference; P<0.001), BASP1 (40% difference; P<0.05) and LAMP (22% difference; P<0.01)) and BPD (STXBP1 (31% difference; P<0.001), BASP1 (23% difference; P<0.01) and LAMP (20% difference; P<0.01)) in the Stanley brain series (n=20 per group). Further validation in dlpfc from the Harvard brain subseries (n=10 per group) confirmed increased protein expression in SCZ of STXBP1 (18% difference; P<0.0001), BASP1 (14% difference; P<0.0001) but not LAMP (20% difference; P=0.14). No significant differences in STXBP1, BASP1 or LAMP protein expression in BPD dlpfc were observed. This study, through proteomic assessments of MM in dlpfc and validation in two brain series, strongly implicates LAMP, STXBP1 and BASP1 in SCZ and supports

  5. Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of cereal yellow dwarf virus.

    Directory of Open Access Journals (Sweden)

    Cecilia Tamborindeguy

    Full Text Available Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV. The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S. graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate

  6. The Effect of Ambient Temperatures of Two Threatened Caribbean Coral Species: a Proteomic Study

    Science.gov (United States)

    Ricaurte, M.; Schizas, N. V.; Weil, E.; Ciborowski, P.; Boukli, N. M.

    2016-02-01

    Coral reefs are among the most valuable ecosystems on the earth. Increasing water temperatures as a consequence of global warming have been identified, as an overriding cause of coral decline (e.g. increased incidence of diseases, bleaching), and one of the regions that has been identified vulnerable to climatic changes, is the Caribbean. Laboratory experiments have shown negative effects of different temperatures in coral growth, larval and adult survival, and gene expression. In order to understand the molecular and cellular basis in the protein regulation during changes in temperature in the field, a comparative proteomic analysis associated with thermal fluctuations was made from wet and dry season of 2014. In the study, we investigated alterations in proteins of Acropora palmata and Orbicella faveolata by two-dimensional gel electrophoresis (2D-GE) followed by liquid chromatography-tandem mass spectrometry, protein identification, and confirmation at the gene expression level by qRT-PCR.Proteomes of related samples demonstrated 195 differentially expressed proteins (DEP) in A. palmata during dry season and 108 (DEP) during wet season of 2014. O. faveolata overexpressed 62 (DEP) in dry season and 190 (DEP) during wet season of 2014. All proteins had a two-fold or greater change in expression due to temperature, altering several components of the cellular stress response that include chaperones, stress proteins, antioxidant enzymes, proteases, cytoskeletal and apoptosis regulating proteins. Our results suggest that A. palmata and O. faveolata display a distinct response by expressing these key protein signatures in dry and wet season. This proteomic approach may open new avenues of research to detect potential early biomarkers involved in response to these stressors, during seasonal changes in water temperatures. The results provide insight into targets and mechanistic strategies to detect potential markers involved in response to temperature change for A

  7. Comparative proteomics reveals that YK51, a 4-Hydroxypandurantin-A analogue, downregulates the expression of proteins associated with dengue virus infection

    Directory of Open Access Journals (Sweden)

    Wei-Lian Tan

    2018-01-01

    Full Text Available Dengue is endemic throughout tropical and subtropical regions of the world. Currently, there is no clinically approved therapeutic drug available for this acute viral infection. Although the first dengue vaccine Dengvaxia has been approved for use in certain countries, it is limited to those without a previous dengue infection while the safety and efficacy of the vaccine in those elderly and younger children still need to be identified. Therefore, it is becoming increasingly important to develop therapeutics/drugs to combat dengue virus (DENV infection. YK51 is a synthetic analogue of 4-Hydroxypandurantin A (a compound found in the crude extract of the rhizomes of Boesenbergia rotunda that has been extensively studied by our research group. It has been shown to possess outstanding antiviral activity due to its inhibitory activity against NS2B/NS3 DENV2 protease. However, it is not known how YK51 affects the proteome of DENV infected cells. Therefore, we performed a comparative proteomics analysis to identify changes in protein expression in DENV infected HepG2 cells treated with YK51. Classical two-dimensional gel electrophoresis followed by protein identification using tandem mass spectrometry was employed in this study. Thirty proteins were found to be down-regulated with YK51 treatment. In silico analysis predicted that the down-regulation of eight of these proteins may inhibit viral infection. Our results suggested that apart from inhibiting the NS2B/NS3 DENV2 protease, YK51 may also be causing the down-regulation of a number of proteins that may be responsible in, and/or essential to virus infection. However, functional characterization of these proteins will be necessary before we can conclusively determine their roles in DENV infection.

  8. Comparative proteomics reveals that YK51, a 4-Hydroxypandurantin-A analogue, downregulates the expression of proteins associated with dengue virus infection.

    Science.gov (United States)

    Tan, Wei-Lian; Lee, Yean Kee; Ho, Yen Fong; Yusof, Rohana; Abdul Rahman, Noorsaadah; Karsani, Saiful Anuar

    2018-01-01

    Dengue is endemic throughout