WorldWideScience

Sample records for chromatin proteome reveals

  1. 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

    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...

  2. Proteomic interrogation of human chromatin.

    Directory of Open Access Journals (Sweden)

    Mariana P Torrente

    Full Text Available Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the "Chromatome" is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes.

  3. Proteomic analyses reveal distinct chromatin-associated and soluble transcription factor complexes.

    Science.gov (United States)

    Li, Xu; Wang, Wenqi; Wang, Jiadong; Malovannaya, Anna; Xi, Yuanxin; Li, Wei; Guerra, Rudy; Hawke, David H; Qin, Jun; Chen, Junjie

    2015-01-21

    The current knowledge on how transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated by protein-protein interactions remains limited. Here, we performed proteomics analyses of soluble and chromatin-associated complexes of 56 TFs, including the targets of many signalling pathways involved in development and cancer, and 37 members of the Forkhead box (FOX) TF family. Using tandem affinity purification followed by mass spectrometry (TAP/MS), we performed 214 purifications and identified 2,156 high-confident protein-protein interactions. We found that most TFs form very distinct protein complexes on and off chromatin. Using this data set, we categorized the transcription-related or unrelated regulators for general or specific TFs. Our study offers a valuable resource of protein-protein interaction networks for a large number of TFs and underscores the general principle that TFs form distinct location-specific protein complexes that are associated with the different regulation and diverse functions of these TFs.

  4. Proteomics of a fuzzy organelle: interphase chromatin

    Science.gov (United States)

    Kustatscher, Georg; Hégarat, Nadia; Wills, Karen L H; Furlan, Cristina; Bukowski-Wills, Jimi-Carlo; Hochegger, Helfrid; Rappsilber, Juri

    2014-01-01

    Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology. PMID:24534090

  5. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.

    Science.gov (United States)

    Smits, Arne H; Jansen, Pascal W T C; Poser, Ina; Hyman, Anthony A; Vermeulen, Michiel

    2013-01-07

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

  6. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components

    DEFF Research Database (Denmark)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Po

    2014-01-01

    To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics durin...

  7. Chromatin Dynamics During DNA Replication and Uncharacterized Replication Factors determined by Nascent Chromatin Capture (NCC) Proteomics

    Science.gov (United States)

    Alabert, Constance; Bukowski-Wills, Jimi-Carlo; Lee, Sung-Bau; Kustatscher, Georg; Nakamura, Kyosuke; de Lima Alves, Flavia; Menard, Patrice; Mejlvang, Jakob; Rappsilber, Juri; Groth, Anja

    2014-01-01

    SUMMARY To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use Nascent Chromatin Capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity-purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3995 proteins. The replication machinery and 485 chromatin factors like CAF-1, DNMT1, SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, while H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance. PMID:24561620

  8. Proteomics and the genetics of sperm chromatin condensation

    Institute of Scientific and Technical Information of China (English)

    Rafael Oliva; Judit Castillo

    2011-01-01

    Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies.

  9. Mass Spectrometry-Based Proteomics for the Analysis of Chromatin Structure and Dynamics

    Directory of Open Access Journals (Sweden)

    Monica Soldi

    2013-03-01

    Full Text Available Chromatin is a highly structured nucleoprotein complex made of histone proteins and DNA that controls nearly all DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, post-translational modifications on histones (hPTMs and DNA methylation, which act in a concerted manner to enforce a specific “chromatin landscape”, with a regulatory effect on gene expression. Mass Spectrometry (MS has emerged as a powerful analytical strategy to detect histone PTMs, revealing interplays between neighbouring PTMs and enabling screens for their readers in a comprehensive and quantitative fashion. Here we provide an overview of the recent achievements of state-of-the-art mass spectrometry-based proteomics for the detailed qualitative and quantitative characterization of histone post-translational modifications, histone variants, and global interactomes at specific chromatin regions. This synopsis emphasizes how the advances in high resolution MS, from “Bottom Up” to “Top Down” analysis, together with the uptake of quantitative proteomics methods by chromatin biologists, have made MS a well-established method in the epigenetics field, enabling the acquisition of original information, highly complementary to that offered by more conventional, antibody-based, assays.

  10. Isolation of Chromatin from Dysfunctional Telomeres Reveals an Important Role for Ring1b in NHEJ-Mediated Chromosome Fusions

    Directory of Open Access Journals (Sweden)

    Cristina Bartocci

    2014-05-01

    Full Text Available When telomeres become critically short, DNA damage response factors are recruited at chromosome ends, initiating a cellular response to DNA damage. We performed proteomic isolation of chromatin fragments (PICh in order to define changes in chromatin composition that occur upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor TRF2. This unbiased purification of telomere-associated proteins in functional or dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at telomeres upon DNA damage induction. On the basis of our results, we describe a critical role for the polycomb group protein Ring1b in nonhomologous end-joining (NHEJ-mediated end-to-end chromosome fusions. We show that cells with reduced levels of Ring1b have a reduced ability to repair uncapped telomeric chromatin. Our data represent an unbiased isolation of chromatin undergoing DNA damage and are a valuable resource to map the changes in chromatin composition in response to DNA damage activation.

  11. Mitochondrial specialization revealed by single muscle fiber proteomics

    DEFF Research Database (Denmark)

    Schiaffino, S; Reggiani, C; Kostrominova, T Y

    2015-01-01

    We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle ...... scavenging capacity to cope with the higher levels of reactive oxygen species production....

  12. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

  13. Quantitative proteomics reveals cellular targets of celastrol.

    Directory of Open Access Journals (Sweden)

    Jakob Hansen

    Full Text Available Celastrol, a natural substance isolated from plant extracts used in traditional Chinese medicine, has been extensively investigated as a possible drug for treatment of cancer, autoimmune diseases, and protein misfolding disorders. Although studies focusing on celastrol's effects in specific cellular pathways have revealed a considerable number of targets in a diverse array of in vitro models there is an essential need for investigations that can provide a global view of its effects. To assess cellular effects of celastrol and to identify target proteins as biomarkers for monitoring treatment regimes, we performed large-scale quantitative proteomics in cultured human lymphoblastoid cells, a cell type that can be readily prepared from human blood samples. Celastrol substantially modified the proteome composition and 158 of the close to 1800 proteins with robust quantitation showed at least a 1.5 fold change in protein levels. Up-regulated proteins play key roles in cytoprotection with a prominent group involved in quality control and processing of proteins traversing the endoplasmic reticulum. Increased levels of proteins essential for the cellular protection against oxidative stress including heme oxygenase 1, several peroxiredoxins and thioredoxins as well as proteins involved in the control of iron homeostasis were also observed. Specific analysis of the mitochondrial proteome strongly indicated that the mitochondrial association of certain antioxidant defense and apoptosis-regulating proteins increased in cells exposed to celastrol. Analysis of selected mRNA transcripts showed that celastrol activated several different stress response pathways and dose response studies furthermore showed that continuous exposure to sub-micromolar concentrations of celastrol is associated with reduced cellular viability and proliferation. The extensive catalog of regulated proteins presented here identifies numerous cellular effects of celastrol and constitutes

  14. Genome-Wide Association between Transcription Factor Expression and Chromatin Accessibility Reveals Regulators of Chromatin Accessibility

    Science.gov (United States)

    Rueedi, Rico

    2017-01-01

    To better understand genome regulation, it is important to uncover the role of transcription factors in the process of chromatin structure establishment and maintenance. Here we present a data-driven approach to systematically characterise transcription factors that are relevant for this process. Our method uses a linear mixed modelling approach to combine datasets of transcription factor binding motif enrichments in open chromatin and gene expression across the same set of cell lines. Applying this approach to the ENCODE dataset, we confirm already known and imply numerous novel transcription factors that play a role in the establishment or maintenance of open chromatin. In particular, our approach rediscovers many factors that have been annotated as pioneer factors. PMID:28118358

  15. 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...... evaluates and monitors intervention in metabolic diseases....

  16. Proteomic Properties Reveal Phyloecological Clusters of Archaea

    Science.gov (United States)

    Nikolic, Nela; Smole, Zlatko; Krisko, Anita

    2012-01-01

    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. PMID:23133575

  17. Proteomic properties reveal phyloecological clusters of Archaea.

    Directory of Open Access Journals (Sweden)

    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.

  18. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    Science.gov (United States)

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

  19. NeuCode Proteomics Reveals Bap1 Regulation of Metabolism

    Directory of Open Access Journals (Sweden)

    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.

  20. Metabolomics reveals a role for the chromatin-binding protein HMGN5 in glutathione metabolism.

    Directory of Open Access Journals (Sweden)

    Eric D Ciappio

    Full Text Available High mobility group nucleosome-binding protein 5 (HMGN5 is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5(+/Y, mice with a targeted mutation in the HMGN5 gene (Hmgn5(tm1/Y, had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5(tm1/Y and their Hmgn5(+/Y littermates. Hmgn5(tm1/Y mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6 and hexokinase 1 (Hk1, was significantly decreased in Hmgn5(tm1/Y mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5(tm1/Y mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.

  1. Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency

    Institute of Scientific and Technical Information of China (English)

    R David Hawkins; Zhen Ye; Samantha Kuan; Pengzhi Yu; Hui Liu; Xinmin Zhang; Roland D Green; Victor V Lobanenkov; Ron Stewart; James A Thomson; Bing Ren; Gary C Hon; Chuhu Yang; Jessica E Antosiewicz-Bourget; LeonardKLee; Que-Minh Ngo; Sarit Klugman; Keith A Ching; Lee E Edsall

    2011-01-01

    Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells.While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types.In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genomewide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage.We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes.We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4mel and H3K27ac,which correlate with expression of their potential target genes.Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs.Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

  2. Diatom proteomics reveals unique acclimation strategies to mitigate Fe limitation.

    Directory of Open Access Journals (Sweden)

    Brook L Nunn

    Full Text Available Phytoplankton growth rates are limited by the supply of iron (Fe in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (Fv/Fm. Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, Thalassiosira pseudonana, was acclimated (10 generations to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, T. pseudonana up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N, C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when T. pseudonana is

  3. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio

    2015-05-01

    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  4. High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins.

    Science.gov (United States)

    Comoglio, Federico; Schlumpf, Tommy; Schmid, Virginia; Rohs, Remo; Beisel, Christian; Paro, Renato

    2015-05-05

    At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  5. Temporal changes in milk proteomes reveal developing milk functions.

    Science.gov (United States)

    Gao, Xinliu; McMahon, Robert J; Woo, Jessica G; Davidson, Barbara S; Morrow, Ardythe L; Zhang, Qiang

    2012-07-06

    Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.

  6. Diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Nicolas M Berbenetz

    2010-09-01

    Full Text Available Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers positions nucleosomes adjacent to the origin to promote replication origin function.

  7. Cassava root membrane proteome reveals activities during storage root maturation.

    Science.gov (United States)

    Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

    2016-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava.

  8. Chromatin landscaping in algae reveals novel regulation pathway for biofuels production

    Energy Technology Data Exchange (ETDEWEB)

    Ngan, Chew Yee; Wong, Chee-Hong; Choi, Cindy; Pratap, Abhishek; Han, James; Wei, Chia-Lin

    2013-02-19

    The diminishing reserve of fossil fuels calls for the development of biofuels. Biofuels are produced from renewable resources, including photosynthetic organisms, generating clean energy. Microalgae is one of the potential feedstock for biofuels production. It grows easily even in waste water, and poses no competition to agricultural crops for arable land. However, little is known about the algae lipid biosynthetic regulatory mechanisms. Most studies relied on the homology to other plant model organisms, in particular Arabidopsis or through low coverage expression analysis to identify key enzymes. This limits the discovery of new components in the biosynthetic pathways, particularly the genetic regulators and effort to maximize the production efficiency of algal biofuels. Here we report an unprecedented and de novo approach to dissect the algal lipid pathways through disclosing the temporal regulations of chromatin states during lipid biosynthesis. We have generated genome wide chromatin maps in chlamydomonas genome using ChIP-seq targeting 7 histone modifications and RNA polymerase II in a time-series manner throughout conditions activating lipid biosynthesis. To our surprise, the combinatory profiles of histone codes uncovered new regulatory mechanism in gene expression in algae. Coupled with matched RNA-seq data, chromatin changes revealed potential novel regulators and candidate genes involved in the activation of lipid accumulations. Genetic perturbation on these candidate regulators further demonstrated the potential to manipulate the regulatory cascade for lipid synthesis efficiency. Exploring epigenetic landscape in microalgae shown here provides powerful tools needed in improving biofuel production and new technology platform for renewable energy generation, global carbon management, and environmental survey.

  9. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics of regulatory sequences.

    Science.gov (United States)

    Kensche, Philip Reiner; Hoeijmakers, Wieteke Anna Maria; Toenhake, Christa Geeke; Bras, Maaike; Chappell, Lia; Berriman, Matthew; Bártfai, Richárd

    2016-03-18

    In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.

  10. Recent advances in maize nuclear proteomic studies reveal histone modifications.

    Science.gov (United States)

    Casati, Paula

    2012-01-01

    The nucleus of eukaryotic organisms is highly dynamic and complex, containing different types of macromolecules including DNA, RNA, and a wide range of proteins. Novel proteomic applications have led to a better overall determination of nucleus protein content. Although nuclear plant proteomics is only at the initial phase, several studies have been reported and are summarized in this review using different plants species, such as Arabidopsis thaliana, rice, cowpea, onion, garden cress, and barrel clover. These include the description of the total nuclear or phospho-proteome (i.e., Arabidopsis, cowpea, onion), or the analysis of the differential nuclear proteome under different growth environments (i.e., Arabidopsis, rice, cowpea, onion, garden cress, and barrel clover). However, only few reports exist on the analysis of the maize nuclear proteome or its changes under various conditions. This review will present recent data on the study of the nuclear maize proteome, including the analysis of changes in posttranslational modifications in histone proteins.

  11. Genomic and proteomic characterization of ARID1A chromatin remodeller in ampullary tumors

    Science.gov (United States)

    Nastase, Anca; Teo, Jin Yao; Heng, Hong Lee; Ng, Cedric Chuan Young; Myint, Swe Swe; Rajasegaran, Vikneswari; Loh, Jia Liang; Lee, Ser Yee; Ooi, London Lucien; Chung, Alexander Yaw Fui; Chow, Pierce Kah Hoe; Cheow, Peng Chung; Wan, Wei Keat; Azhar, Rafy; Khoo, Avery; Xiu, Sam Xin; Alkaff, Syed Muhammad Fahmy; Cutcutache, Ioana; Lim, Jing Quan; Ong, Choon Kiat; Herlea, Vlad; Dima, Simona; Duda, Dan G; Teh, Bin Tean; Popescu, Irinel; Lim, Tony Kiat Hon

    2017-01-01

    AT rich interactive domain 1A (ARID1A) is one of the most commonly mutated genes in a broad variety of tumors. The mechanisms that involve ARID1A in ampullary cancer progression remains elusive. Here, we evaluated the frequency of ARID1A and KRAS mutations in ampullary adenomas and adenocarcinomas and in duodenal adenocarcinomas from two cohorts of patients from Singapore and Romania, correlated with clinical and pathological tumor features, and assessed the functional role of ARID1A. In the ampullary adenocarcinomas, the frequency of KRAS and ARID1A mutations was 34.7% and 8.2% respectively, with a loss or reduction of ARID1A protein in 17.2% of the cases. ARID1A mutational status was significantly correlated with ARID1A protein expression level (P=0.023). There was a significant difference in frequency of ARID1A mutation between Romania and Singapore (2.7% versus 25%, P=0.04), suggestive of different etiologies. One somatic mutation was detected in the ampullary adenoma group. In vitro studies indicated the tumor suppressive role of ARID1A. Our results warrant further investigation of this chromatin remodeller as a potential early biomarker of the disease, as well as identification of therapeutic targets in ARID1A mutated ampullary cancers.

  12. 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

    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......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...... 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...

  13. 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...... 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...

  14. 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 ...... evaluates and monitors intervention in metabolic diseases....... in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly...

  15. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.

    Directory of Open Access Journals (Sweden)

    Harm van Bakel

    2013-05-01

    Full Text Available Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

  16. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.

    Science.gov (United States)

    van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R; Nislow, Corey

    2013-05-01

    Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

  17. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Directory of Open Access Journals (Sweden)

    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.

  18. Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes

    Science.gov (United States)

    Hua, Qingzhu; Zhou, Qianjun; Gan, Susheng; Wu, Jingyu; Chen, Canbin; Li, Jiaqiang; Ye, Yaoxiong; Zhao, Jietang; Hu, Guibing; Qin, Yonghua

    2016-01-01

    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. PMID:27690004

  19. Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

    Science.gov (United States)

    Liu, Jing; Vidi, Pierre-Alexandre; Lelièvre, Sophie A; Irudayaraj, Joseph M K

    2015-02-01

    Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

  20. Proteomic analyses reveal divergent ubiquitylation site patterns in murinetissues

    DEFF Research Database (Denmark)

    Wagner, Sebastian A; Beli, Petra; Weinert, Brian T;

    2012-01-01

    Posttranslational modifications of proteins increase the complexity of the cellular proteome andenable rapid regulation of protein functions in response to environmental changes. Proteinubiquitylation is a central regulatory posttranslational modification that controls numerousbiological processes...

  1. Recent advances in maize nuclear proteomic studies reveal histone modifications

    Directory of Open Access Journals (Sweden)

    Paula eCasati

    2012-12-01

    Full Text Available The nucleus of eukaryotic organisms is highly dynamic and complex, containing different types of macromolecules including DNA, RNA, and a wide range of proteins. Novel proteomic applications have led to a better overall determination of nucleus protein content. Although nuclear plant proteomics is only at the initial phase, several studies have been reported and are summarized in this review using different plants species, such as Arabidopsis thaliana, rice, cowpea, onion, garden cress, and barrel clover. These include the description of the total nuclear or phospho-proteome (i.e. Arabidopsis, cowpea, onion, or the analysis of the differential nuclear proteome under different growth environments (i.e. Arabidopsis, rice, cowpea, onion, garden cress and barrel clover. However, only few reports exist on the analysis of the maize nuclear proteome or its changes under various conditions. This review will present recent data on the study of the nuclear maize proteome, including the analysis of changes in posttranslational modifications in histone proteins.

  2. Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

    Science.gov (United States)

    Wachter, Elisabeth; Quante, Timo; Merusi, Cara; Arczewska, Aleksandra; Stewart, Francis; Webb, Shaun; Bird, Adrian

    2014-01-01

    The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs. DOI: http://dx.doi.org/10.7554/eLife.03397.001 PMID:25259796

  3. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  4. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    Science.gov (United States)

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

  5. Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin.

    Directory of Open Access Journals (Sweden)

    Alexandre Amlie-Wolf

    Full Text Available TAR DNA-binding protein 43 (TDP-43 is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP, TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43 so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3' untranslated region (UTR processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains.

  6. Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

    Science.gov (United States)

    Vera, Daniel L; Madzima, Thelma F; Labonne, Jonathan D; Alam, Mohammad P; Hoffman, Gregg G; Girimurugan, S B; Zhang, Jinfeng; McGinnis, Karen M; Dennis, Jonathan H; Bass, Hank W

    2014-10-01

    The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5' and 3' ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

  7. Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

    Science.gov (United States)

    Thrower, D A; Bloom, K

    2001-09-01

    We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

  8. Quantitative Proteomics of Intracellular Campylobacter jejuni Reveals Metabolic Reprogramming.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Liu

    Full Text Available Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.

  9. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription.

    Science.gov (United States)

    Tang, Zhonghui; Luo, Oscar Junhong; Li, Xingwang; Zheng, Meizhen; Zhu, Jacqueline Jufen; Szalaj, Przemyslaw; Trzaskoma, Pawel; Magalska, Adriana; Wlodarczyk, Jakub; Ruszczycki, Blazej; Michalski, Paul; Piecuch, Emaly; Wang, Ping; Wang, Danjuan; Tian, Simon Zhongyuan; Penrad-Mobayed, May; Sachs, Laurent M; Ruan, Xiaoan; Wei, Chia-Lin; Liu, Edison T; Wilczynski, Grzegorz M; Plewczynski, Dariusz; Li, Guoliang; Ruan, Yijun

    2015-12-17

    Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

  10. Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Directory of Open Access Journals (Sweden)

    Sha Ky

    2010-08-01

    Full Text Available Abstract Background Tissue differentiation is accompanied by genome-wide changes in the underlying chromatin structure and dynamics, or epigenome. By controlling when, where, and what regulatory factors have access to the underlying genomic DNA, the epigenome influences the cell's transcriptome and ultimately its function. Existing genomic methods for analyzing cell-type-specific changes in chromatin generally involve two elements: (i a source for purified cells (or nuclei of distinct types, and (ii a specific treatment that partitions or degrades chromatin by activity or structural features. For many cell types of great interest, such assays are limited by our inability to isolate the relevant cell populations in an organism or complex tissue containing an intertwined mixture of other cells. This limitation has confined available knowledge of chromatin dynamics to a narrow range of biological systems (cell types that can be sorted/separated/dissected in large numbers and tissue culture models or to amalgamations of diverse cell types (tissue chunks, whole organisms. Results Transgene-driven expression of DNA/chromatin modifying enzymes provides one opportunity to query chromatin structures in expression-defined cell subsets. In this work we combine in vivo expression of a bacterial DNA adenine methyltransferase (DAM with high throughput sequencing to sample tissue-specific chromatin accessibility on a genome-wide scale. We have applied the method (DALEC: Direct Asymmetric Ligation End Capture towards mapping a cell-type-specific view of genome accessibility as a function of differentiated state. Taking advantage of C. elegans strains expressing the DAM enzyme in diverse tissues (body wall muscle, gut, and hypodermis, our efforts yield a genome-wide dataset measuring chromatin accessibility at each of 538,000 DAM target sites in the C. elegans (diploid genome. Conclusions Validating the DALEC mapping results, we observe a strong association

  11. Global analysis of SUMO chain function reveals multiple roles in chromatin regulation.

    Science.gov (United States)

    Srikumar, Tharan; Lewicki, Megan C; Costanzo, Michael; Tkach, Johnny M; van Bakel, Harm; Tsui, Kyle; Johnson, Erica S; Brown, Grant W; Andrews, Brenda J; Boone, Charles; Giaever, Guri; Nislow, Corey; Raught, Brian

    2013-04-01

    Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric "chains," but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3(allR)) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3(allR) cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a >1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast.

  12. Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

    Science.gov (United States)

    Pérez-Lluch, Sílvia; Blanco, Enrique; Carbonell, Albert; Raha, Debasish; Snyder, Michael; Serras, Florenci; Corominas, Montserrat

    2011-06-01

    An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

  13. Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

    DEFF Research Database (Denmark)

    Grassl, Niklas; Kulak, Nils Alexander; Pichler, Garwin

    2016-01-01

    , disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow. METHODS: We used...... with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. CONCLUSION: Rapid shotgun...... and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host-pathogen interactions and clinical saliva diagnostics....

  14. Proteomics

    DEFF Research Database (Denmark)

    Dam, Svend; Stougaard, Jens

    2014-01-01

    proteomics data. Two characteristics of legumes are the high seed protein level and the nitrogen fixing symbiosis. Thus, the majority of the proteomics studies in Lotus have been performed on seed/pod and nodule/root tissues in order to create proteome reference maps and to enable comparative analyses within...... Lotus tissues or toward similar tissues from other legume species. More recently, N-glycan structures and compositions have been determined from mature Lotus seeds using glycomics and glycoproteomics, and finally, phosphoproteomics has been employed...... and annotated Lotus japonicus (Lotus) genome has been essential for obtaining high-quality protein identifications from proteomics studies. Furthermore, additional genomics and transcriptomics studies from several Lotus species/ecotypes support putative gene structures and these can be further supported using...

  15. Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis thaliana.

    Science.gov (United States)

    van Drunen, C M; Oosterling, R W; Keultjes, G M; Weisbeek, P J; van Driel, R; Smeekens, S C

    1997-10-01

    The Arabidopsis thaliana genome is currently being sequenced, eventually leading towards the unravelling of all potential genes. We wanted to gain more insight into the way this genome might be organized at the ultrastructural level. To this extent we identified matrix attachment regions demarking potential chromatin domains, in a 16 kb region around the plastocyanin gene. The region was cloned and sequenced revealing six genes in addition to the plastocyanin gene. Using an heterologous in vitro nuclear matrix binding assay, to search for evolutionary conserved matrix attachment regions (MARs), we identified three such MARs. These three MARs divide the region into two small chromatin domains of 5 kb, each containing two genes. Comparison of the sequence of the three MARs revealed a degenerated 21 bp sequence that is shared between these MARs and that is not found elsewhere in the region. A similar sequence element is also present in four other MARs of Arabidopsis.Therefore, this sequence may constitute a landmark for the position of MARs in the genome of this plant. In a genomic sequence database of Arabidopsis the 21 bp element is found approximately once every 10 kb. The compactness of the Arabidopsis genome could account for the high incidence of MARs and MRSs we observed.

  16. Yeast genetic analysis reveals the involvement of chromatin reassembly factors in repressing HIV-1 basal transcription.

    Directory of Open Access Journals (Sweden)

    Manuela Vanti

    2009-01-01

    Full Text Available Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5' end of HIV-1 transcribed region (5'HIV-TR, which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5'HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5'HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5'HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.

  17. Molecular Mechanisms of Processing Proteome Reorganization of Interphase Chromatin During Stress and Adaptation to Winter in Wheat

    Directory of Open Access Journals (Sweden)

    Ivanov R.S.

    2015-06-01

    Full Text Available Research of fundamental molecular and genetic processes of plant interaction with the environment, is a progressive field of understanding the fundamental problems of stress supramolecular biochemistry of developmental biology. The purpose of the work was the analysis of localization shielded to protease processing proteins of suprastructures of interphase chromatin matrix in the conditions of adaptation during vegetative phase of wheat to stressful environment factors. It is shown that in the conditions of perennial adaptation to cold shock of wheat at the level of chromatin suprastructures tightly bound to the nuclear matrix there is a total shielding of arginine-X sites to protease-processing. Perhaps these are zones that affect to the architecture organization of the cell nucleus that can help to survive in complex environmental conditions. According to the priorities in the study of agricultural plants, put forward by EPIC (The Epigenomics of Plants International Consortium in 2011 for the next decade, was included the point of necessity to understand the molecular basis of the interactions of genotype and environment that change the characteristics of plants in different conditions of the environment. These data will be useful for those who involved in the development of mathematical logic schemes of the theory and practice of biological specificity, and it could be included in the ontology of the stages plant growth and development.

  18. 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.

  19. Proteomics of differential extraction fractions enriched for chromatin-binding proteins from colon adenoma and carcinoma tissues

    DEFF Research Database (Denmark)

    Knol, Jaco C; de Wit, Meike; Albrethsen, Jakob

    2014-01-01

    of colorectal cancer (CRC) progression. METHODS: CB protein-containing fractions were biochemically extracted from human colorectal tissues, including carcinomas with chromosomal instability (CIN), carcinomas with microsatellite instability (MIN), and adenomas. The CB proteins were subjected to label-free LC...... increased, whereas specific components of chromosome segregation and DNA recombination/repair systems were decreased. CONCLUSIONS: Our study identifies proteomic changes at the subnuclear level that are associated with CRC and may be further investigated. This article is part of a Special Issue entitled...

  20. 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

    architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts......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...

  1. Plasma proteome response to severe burn injury revealed by 18O-labeled "universal" reference-based quantitative proteomics.

    Science.gov (United States)

    Qian, Wei-Jun; Petritis, Brianne O; Kaushal, Amit; Finnerty, Celeste C; Jeschke, Marc G; Monroe, Matthew E; Moore, Ronald J; Schepmoes, Athena A; Xiao, Wenzhong; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2010-09-01

    A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of approximately 35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

  2. High-Resolution Mapping of Protein Concentration Reveals Principles of Proteome Architecture and Adaptation

    Directory of Open Access Journals (Sweden)

    Emmanuel D. Levy

    2014-05-01

    Full Text Available A single yeast cell contains a hundred million protein molecules. How these proteins are organized to orchestrate living processes is a central question in biology. To probe this organization in vivo, we measured the local concentration of proteins based on the strength of their nonspecific interactions with a neutral reporter protein. We first used a cytosolic reporter and measured local concentrations for ∼2,000 proteins in S. cerevisiae, with accuracy comparable to that of mass spectrometry. Localizing the reporter to membranes specifically increased the local concentration measured for membrane proteins. Comparing the concentrations measured by both reporters revealed that encounter frequencies between proteins are primarily dictated by their abundances. However, to change these encounter frequencies and restructure the proteome, as in adaptation, we find that changes in localization have more impact than changes in abundance. These results highlight how protein abundance and localization contribute to proteome organization and reorganization.

  3. Mitochondrial specialization revealed by single muscle fiber proteomics: focus on the Krebs cycle.

    Science.gov (United States)

    Schiaffino, S; Reggiani, C; Kostrominova, T Y; Mann, M; Murgia, M

    2015-12-01

    We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

  4. Temporal proteomic analysis reveals continuous impairment of intestinal development in neonatal piglets with intrauterine growth restriction.

    Science.gov (United States)

    Wang, Xiaoqiu; Wu, Weizong; Lin, Gang; Li, Defa; Wu, Guoyao; Wang, Junjun

    2010-02-01

    Efficiency of nutrient utilization is reduced in neonates with intrauterine growth restriction (IUGR) compared with those with a normal birth weight (NBW). However, the underlying mechanisms are largely unknown. In this study, we applied temporal proteomic approach, coupled with histological and biochemical analyses, to study dynamic changes of the proteome in the small intestinal mucosa of IUGR piglets during the nursing period (Days 1, 7 and 21). We identified 56 differentially expressed protein spots between IUGR and NBW piglets. These proteins participate in key biological processes, including (1) absorption, digestion and transport of nutrients; (2) cell structure and motility; (3) glucose and energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitamin metabolism; (7) cellular redox homeostasis; (8) stress response; and (9) apoptosis. The results of our temporal proteomics analysis reveal continuous impairment of intestinal development in neonatal piglets with IUGR. The findings have important implications for understanding metabolic defects in the small intestine of IUGR neonates and are expected to provide new strategies to improve their survival and growth.

  5. Proteomics

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...... grouped manually to one or more of five major functional groups related to metabolism, cell structure, immunity, apoptosis and angiogenesis. These were chosen to represent basic cell functions and biological processes potentially involved in the pathogenesis of CHD. The LC–MS/MS-based proteomic analysis...

  6. Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

    Science.gov (United States)

    Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raibatak; Kokotovic, Marko; Grimm, Jonathan B; Lavis, Luke D; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

    2016-01-01

    The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes. DOI: http://dx.doi.org/10.7554/eLife.17667.001 PMID:27723458

  7. Proteomic analysis reveals novel proteins associated with progression and differentiation of colorectal carcinoma

    Directory of Open Access Journals (Sweden)

    Yi Gan

    2014-01-01

    Full Text Available Aim: The objective of this study is to characterize differential proteomic expression among well-differentiation and poor-differentiation colorectal carcinoma tissues and normal mucous epithelium. Materials and Methods: The study is based on quantitative 2-dimensional gel electrophoresis and analyzed by PDquest. Results: Excluding redundancies due to proteolysis and posttranslational modified isoforms of over 600 protein spots, 11 proteins were revealed as regulated with statistical variance being within the 95 th confidence level and were identified by peptide mass fingerprinting in matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Progression-associated proteins belong to the functional complexes of tumorigenesis, proliferation, differentiation, metabolism, and the regulation of major histocompatibility complex processing and other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in CRC. Among various differentiation stage of CRC tissues, we identified calreticulin precursor, MHC class I antigen (human leukocyte antigen A , glutathione S-transferase pi1, keratin 8, heat shock protein 27, tubulin beta chain, triosephosphate, fatty acid-binding protein, hemoglobin (deoxy mutant with val b 1 replaced by met (HBB, and zinc finger protein 312 (FEZF2. Conclusions: Their functional networks were analyzed by Ingenuity systems Ingenuity Pathways Analysis and revealed the potential roles as novel biomarkers for progression in various differentiation stages of CRC.

  8. Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins

    Science.gov (United States)

    CRUZ*, ISA N.; COLEY*, HELEN M.; KRAMER, HOLGER B.; MADHURI, THUMULURU KAVITAH; SAFUWAN, NUR A.M.; ANGELINO, ANA RITA; YANG, MIN

    2016-01-01

    Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. Materials and Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer. *These Authors contributed equally to this study. PMID:28031236

  9. Reprogramming chromatin

    DEFF Research Database (Denmark)

    Ehrensberger, Andreas Hasso; Svejstrup, Jesper Qualmann

    2012-01-01

    attributed to high kinetic barriers that affect all cells equally and can only be overcome by rare stochastic events. The barriers to reprogramming are likely to involve transformations of chromatin state because (i) inhibitors of chromatin-modifying enzymes can enhance the efficiency of reprogramming...... and (ii) knockdown or knock-out of chromatin-modifying enzymes can lower the efficiency of reprogramming. Here, we review the relationship between chromatin state transformations (chromatin reprogramming) and cellular reprogramming, with an emphasis on transcription factors, chromatin remodeling factors...

  10. A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors

    Directory of Open Access Journals (Sweden)

    Lior Izhar

    2015-06-01

    Full Text Available Localization to sites of DNA damage is a hallmark of DNA damage response (DDR proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose polymerase (PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

  11. 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

    2017-01-01

    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. PMID:28119706

  12. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer.

    Science.gov (United States)

    Kim, Jae-Young; Stewart, Paul A; Borne, Adam L; Fang, Bin; Welsh, Eric A; Chen, Yian Ann; Eschrich, Steven A; Koomen, John M; Haura, Eric B

    2016-06-01

    One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  13. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    2016-04-01

    Full Text Available One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  14. 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.

  15. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J [Pacific Northwest National Laboratory (PNNL); Wilkins, Mike [University of California, Berkeley; Nicora, Carrie D. [Pacific Northwest National Laboratory (PNNL); Williams, Ken [Lawrence Berkeley National Laboratory (LBNL); Banfield, Jillian F. [University of California, Berkeley; Verberkmoes, Nathan C [ORNL; Hettich, Robert {Bob} L [ORNL; N' Guessan, A. Lucie [University of Massachusetts, Amherst; Mouser, Paula J [University of Massachusetts, Amherst; Elifantz, Hila [University of Massachusetts, Amherst; Smith, Richard D. [Pacific Northwest National Laboratory (PNNL); Lovley, Derek [University of Massachusetts, Amherst; Lipton, Mary S [Pacific Northwest National Laboratory (PNNL); Long, Phil [Pacific Northwest National Laboratory (PNNL)

    2010-01-01

    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 acetateamended 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,ashift 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. 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...... packing did not lead to distinct changes in protein pattern. Applying DPLSR to the 2-DE data enabled the selection of protein spots critical for differentiation between 3 and 6months frozen storage with 12months frozen storage. Some of these protein spots have been identified by MS/MS, revealing myosin...

  17. 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

    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...... 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...... 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...

  18. 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.

  19. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

    Directory of Open Access Journals (Sweden)

    Athale Chaitanya

    2004-11-01

    Full Text Available Abstract Background The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. Results We developed a novel 4-D image processing platform (TIKAL for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 μm – wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. Conclusions Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M

  20. Proteomic profile of Cryptococcus neoformans biofilm reveals changes in metabolic processes.

    Science.gov (United States)

    Santi, Lucélia; Beys-da-Silva, Walter O; Berger, Markus; Calzolari, Diego; Guimarães, Jorge A; Moresco, James J; Yates, John R

    2014-03-07

    Cryptococcus neoformans, a pathogenic yeast, causes meningoencephalitis, especially in immunocompromised patients, leading in some cases to death. Microbes in biofilms can cause persistent infections, which are harder to treat. Cryptococcal biofilms are becoming common due to the growing use of brain valves and other medical devices. Using shotgun proteomics we determine the differences in protein abundance between biofilm and planktonic cells. Applying bioinformatic tools, we also evaluated the metabolic pathways involved in biofilm maintenance and protein interactions. Our proteomic data suggest general changes in metabolism, protein turnover, and global stress responses. Biofilm cells show an increase in proteins related to oxidation-reduction, proteolysis, and response to stress and a reduction in proteins related to metabolic process, transport, and translation. An increase in pyruvate-utilizing enzymes was detected, suggesting a shift from the TCA cycle to fermentation-derived energy acquisition. Additionally, we assign putative roles to 33 proteins previously categorized as hypothetical. Many changes in metabolic enzymes were identified in studies of bacterial biofilm, potentially revealing a conserved strategy in biofilm lifestyle.

  1. Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics.

    Directory of Open Access Journals (Sweden)

    Takeshi Takeuchi

    Full Text Available Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1 formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2 additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3 calcification controlled by coral-specific SOMPs.

  2. A Shotgun Proteomics Approach Reveals a New Toxic Role For Alzheimer's Disease Aβ Peptide: Spliceosome Impairment.

    Science.gov (United States)

    Nuzzo, Domenico; Inguglia, Luigi; Walters, Jessica; Picone, Pasquale; Di Carlo, Marta

    2017-02-03

    Proteomic changes have been described in many neurodegenerative diseases including Alzheimer's disease (AD). However, the early events in the onset of the pathology are yet to be fully elucidated. A cell model system in which LAN5 neuroblastoma cells were incubated for a short time with a recombinant form of Aβ42 (rAβ42) was utilized. Proteins extracted from these cells were subjected to shotgun proteomics analysis by LTQ-Orbitrap-MS followed by label-free quantitation. By bioinformatics tools we found that the most significant of those found to be up-regulated were related to cytoskeletal dynamics (Rho related) and membrane related processes. The most significant of the down-regulated proteins were hnRNP-related. In particular hnRNPs involved in ribosomal biogenesis and in splicing were down-regulated. The latter of these processes stood out as it was highlighted ubiquitously and with the highest significance in the results of every analysis. Furthermore, our findings revealed downregulation at every stage of the splicing process through down-regulation of every subunit of the spliceosome. Dysregulation of the spliceosome was also confirmed using a Western blot ì In conclusion, these data suggest dysregulation of the proteins and processes identified as early events in pathogenesis of AD following Aβ accumulation.

  3. Comprehensive Proteomics Analysis of Laticifer Latex Reveals New Insights into Ethylene Stimulation of Natural Rubber Production.

    Science.gov (United States)

    Wang, Xuchu; Wang, Dan; Sun, Yong; Yang, Qian; Chang, Lili; Wang, Limin; Meng, Xueru; Huang, Qixing; Jin, Xiang; Tong, Zheng

    2015-09-08

    Ethylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.

  4. Comparative transcriptome and proteome analysis to reveal the biosynthesis of gold nanoparticles in Arabidopsis.

    Science.gov (United States)

    Tiwari, Manish; Krishnamurthy, Sneha; Shukla, Devesh; Kiiskila, Jeffrey; Jain, Ajay; Datta, Rupali; Sharma, Nilesh; Sahi, Shivendra V

    2016-02-23

    A large number of plants have been tested and exploited in search of a green chemistry approach for the fabrication of gold or other precious metal nanomaterials. Despite the potential of plant based methods, very little is known about the underlying biochemical reactions and genes involved in the biotransformation mechanism of AuCl4 into gold nanoparticles (AuNPs). In this research, we thus focused on studying the effect of Au on growth and nanoparticles formation by analyses of transcriptome, proteome and ionome shift in Arabidopsis. Au exposure favored the growth of Arabidopsis seedling and induced formation of nanoparticles in root and shoot, as indicated by optical and hyperspectral imaging. Root transcriptome analysis demonstrated the differential expression of the members of WRKY, MYB and BHLH gene families, which are involved in the Fe and other essential metals homeostasis. The proteome analysis revealed that Glutathione S-transferases were induced in the shoot and suggested its potential role in the biosynthesis AuNPs. This study also demonstrated the role of plant hormone auxin in determining the Au induced root system architecture. This is the first study using an integrated approach to understand the in planta biotransformation of KAuCl4 into AuNPs.

  5. Proteomic analysis of mice fed methionine and choline deficient diet reveals marker proteins associated with steatohepatitis.

    Directory of Open Access Journals (Sweden)

    Su Jin Lee

    Full Text Available The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH. Mice fed a methionine and choline deficient diet (MCD developed hepatic steatosis characterized by increased free fatty acid (FFA and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.

  6. In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

    Directory of Open Access Journals (Sweden)

    Sara Zanivan

    2013-02-01

    Full Text Available Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

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

    Science.gov (United States)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    2015-03-19

    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 architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level.

  8. Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

    Directory of Open Access Journals (Sweden)

    Tang Vivian W

    2006-12-01

    Full Text Available Abstract Background Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of ~100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown. Results A novel biochemical purification protocol has been developed to isolate tight junction protein complexes from cultured human epithelial cells. After identification of proteins by mass spectroscopy and fingerprint analysis, candidate proteins are scored and assessed individually. A simple algorithm has been devised to incorporate transmembrane domains and protein modification sites for scoring membrane proteins. Using this new scoring system, a total of 912 proteins have been identified. These 912 hits are analyzed using a bioinformatics approach to bin the hits in 4 categories: configuration, molecular function, cellular function, and specialized process. Prominent clusters of proteins related to the cytoskeleton, cell adhesion, and vesicular traffic have been identified. Weaker clusters of proteins associated with cell growth, cell migration, translation, and transcription are also found. However, the strongest clusters belong to synaptic proteins and signaling molecules. Localization studies of key components of synaptic transmission have confirmed the presence of both presynaptic and postsynaptic proteins at the tight junction domain. To correlate proteomics data with structure, the tight junction has been examined using electron microscopy. This has revealed many novel structures including end-on cytoskeletal attachments, vesicles fusing/budding at the tight junction membrane domain, secreted substances encased between the tight junction kisses, endocytosis of tight junction

  9. Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus.

    Directory of Open Access Journals (Sweden)

    Jiwu Chen

    Full Text Available In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.

  10. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    Energy Technology Data Exchange (ETDEWEB)

    Callister, Stephen J.; Wilkins, Michael J.; Nicora, Carrie D.; Williams, Kenneth H.; Banfield, Jillian F.; VerBerkmoes, Nathan; Hettich, Robert L.; N' Guessan, A. Lucie; Mouser, Paula; Elifantz, H.; Smith, Richard D.; Lovley, Derek R.; Lipton, Mary S.; Long, Philip E.

    2010-12-01

    Stimulated by acetate-amendment field experiments conducted in 2007 and 2008, 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 period, planktonic biomass was sampled at various time points and used to quantitatively evaluate proteomes, both spatially and temporally to study the dynamics of the microbial community proteome dynamics in relationship to geochemical measurements. As there were no comprehensive genome sequence data available at the time, we systematically evaluated different organisms to generate a "pseudo-metagenome" for proteomics analyses. Proteomics results supported the dominance of Geobacteraceae during biostimulation and revealed a shift from iron reduction to sulfate reduction, evidenced by changes in community membership. Because U(VI) is reduced at a lower rate during sulfate reduction, detecting this shift is important to maintaining the maximum rate of U(VI) reduction. In addition, the comparison of proteome measurements made at the end of the 2007 field experiment to the 2008 field experiment revealed a modified community structure. Importantly, the failure of a community to rebound following the cessation of biostimulation needs to be included in long-term remediation strategies.

  11. Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus.

    Science.gov (United States)

    Kebaabetswe, Lemme P; Haick, Anoria K; Gritsenko, Marina A; Fillmore, Thomas L; Chu, Rosalie K; Purvine, Samuel O; Webb-Robertson, Bobbie-Jo; Matzke, Melissa M; Smith, Richard D; Waters, Katrina M; Metz, Thomas O; Miura, Tanya A

    2015-09-01

    Infection of type II alveolar epithelial (ATII) cells by influenza A viruses (IAV) correlates with severe respiratory disease in humans and mice. To understand pathogenic mechanisms during IAV infection of ATII cells, murine ATII cells were cultured to maintain a differentiated phenotype, infected with IAV-PR8, which causes severe lung pathology in mice, and proteomics analyses were performed using liquid chromatography-mass spectrometry. PR8 infection increased levels of proteins involved in interferon signaling, antigen presentation, and cytoskeleton regulation. Proteins involved in mitochondrial membrane permeability, energy metabolism, and chromatin formation had reduced levels in PR8-infected cells. Phenotypic markers of ATII cells in vivo were identified, confirming the differentiation status of the cultures. Surfactant protein B had decreased levels in PR8-infected cells, which was confirmed by immunoblotting and immunofluorescence assays. Analysis of ATII cell protein profiles will elucidate cellular processes in IAV pathogenesis, which may provide insight into potential therapies to modulate disease severity.

  12. 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.

  13. 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

  14. Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection.

    Directory of Open Access Journals (Sweden)

    Qian Ma

    Full Text Available Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4-6 folds elongated, and formed intracytoplasmic membranes (ICM. To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.

  15. Proteomic and Physiological Analyses Reveal Detoxification and Antioxidation Induced by Cd Stress in Kandelia candel Roots

    Institute of Scientific and Technical Information of China (English)

    Zhaoxia Weng; Lingxia Wang; Fanglin Tan; Li Huang; Jianhong Xing; Shipin Chen; Chilien Cheng

    2012-01-01

    The heavy metal Cadmium (Cd),added to the water bodies through weathering of rocks and human activities,constitutes one of the major environmental pollutants toxic to plants.This study examines the proteome changes in roots of actively growing Kandelia candel (L.) Druce when challenged with Cd.This mangrove-like species proliferates in estuaries and bays and is a potential choice for phytoremediation of Cd.A total of 53 proteins were up-or down-regulated following a short-term Cd treatment.The identities of the differentially expressed proteins were determined by MALDI-TOF/TOF.Approximately half of the up-regulated proteins are involved in oxidative response,including antioxidant enzymes,enzymes required for glutathione biosynthesis,enzymes in TCA and PPP cycles for generating ATP,NADH and NADPH.These results support the prediction that a prompt antioxidative response is necessary for the reduction of the oxidative stress caused by Cd and set the stage for further investigating of Cd up-regulated proteins in Kandelia candel.In summary,this investigation of global proteomic changes in K.candel roots reveals a complex cellular network affected by Cd stress.The network covers a broad range of metabolic processes,including protein synthesis,antioxidative/detoxifying reactions,energy generation,and metabolites production against Cd stress.Particularly important,our results support the predicted key roles of glutathione biosynthesis,ascorbate-glutathione cycle and antioxidative defense system in Cd detoxification in K.candel roots.This understanding is necessary for developing the woody plant K.candel for phytoremediation of Cd and other heavy metals and may be critical for maintaining health mangrove ecology.

  16. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin.

    Science.gov (United States)

    Bancaud, Aurélien; Huet, Sébastien; Daigle, Nathalie; Mozziconacci, Julien; Beaudouin, Joël; Ellenberg, Jan

    2009-12-16

    The nucleus of eukaryotes is organized into functional compartments, the two most prominent being heterochromatin and nucleoli. These structures are highly enriched in DNA, proteins or RNA, and thus thought to be crowded. In vitro, molecular crowding induces volume exclusion, hinders diffusion and enhances association, but whether these effects are relevant in vivo remains unclear. Here, we establish that volume exclusion and diffusive hindrance occur in dense nuclear compartments by probing the diffusive behaviour of inert fluorescent tracers in living cells. We also demonstrate that chromatin-interacting proteins remain transiently trapped in heterochromatin due to crowding induced enhanced affinity. The kinetic signatures of these crowding consequences allow us to derive a fractal model of chromatin organization, which explains why the dynamics of soluble nuclear proteins are affected independently of their size. This model further shows that the fractal architecture differs between heterochromatin and euchromatin, and predicts that chromatin proteins use different target-search strategies in the two compartments. We propose that fractal crowding is a fundamental principle of nuclear organization, particularly of heterochromatin maintenance.

  17. Polymer model with Epigenetic Recoloring Reveals a Pathway for the de novo Establishment and 3D Organization of Chromatin Domains

    Science.gov (United States)

    Michieletto, D.; Orlandini, E.; Marenduzzo, D.

    2016-10-01

    One of the most important problems in development is how epigenetic domains can first be established, and then maintained, within cells. To address this question, we propose a framework that couples three-dimensional chromatin folding dynamics to a "recoloring" process modeling the writing of epigenetic marks. Because many intrachromatin interactions are mediated by bridging proteins, we consider a "two-state" model with self-attractive interactions between two epigenetic marks that are alike (either active or inactive). This model displays a first-order-like transition between a swollen, epigenetically disordered phase and a compact, epigenetically coherent chromatin globule. If the self-attraction strength exceeds a threshold, the chromatin dynamics becomes glassy, and the corresponding interaction network freezes. By modifying the epigenetic read-write process according to more biologically inspired assumptions, our polymer model with recoloring recapitulates the ultrasensitive response of epigenetic switches to perturbations and accounts for long-lived multidomain conformations, strikingly similar to the topologically associating domains observed in eukaryotic chromosomes.

  18. Chromatin computation.

    Directory of Open Access Journals (Sweden)

    Barbara Bryant

    Full Text Available In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this "chromatin computer" to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal--and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines.

  19. Proteome Differences between Hepatitis B Virus Genotype-B- and Genotype-C-Induced Hepatocellular Carcinoma Revealed by iTRAQ-Based Quantitative Proteomics.

    Science.gov (United States)

    Wei, Dahai; Zeng, Yongyi; Xing, Xiaohua; Liu, Hongzhi; Lin, Minjie; Han, Xiao; Liu, Xiaolong; Liu, Jingfeng

    2016-02-05

    Hepatitis B virus (HBV) is the main cause of hepatocellular carcinoma (HCC) in southeast Asia where HBV genotype B and genotype C are the most prevalent. Viral genotypes have been reported to significantly affect the clinical outcomes of HCC. However, the underlying molecular differences among different genotypes of HBV virus infected HCC have not been revealed. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify the proteome differences between the HBV genotypes B- and C-induced HCC. In brief, a total of 83 proteins in the surrounding noncancerous tissues and 136 proteins in the cancerous tissues between HBV genotype-B- and genotype-C-induced HCC were identified, respectively. This information revealed that there might be different molecular mechanisms of the tumorigenesis and development of HBV genotypes B- and C-induced HCC. Furthermore, our results indicate that the two proteins ARFIP2 and ANXA1 might be potential biomarkers for distinguishing the HBV genotypes B- and C-induced HCC. Thus, the quantitative proteomic analysis revealed molecular differences between the HBV genotypes B- and C-induced HCC, and might provide fundamental information for further deep study.

  20. Significant modifications of the salivary proteome potentially associated with complications of Down syndrome revealed by top-down proteomics.

    Science.gov (United States)

    Cabras, Tiziana; Pisano, Elisabetta; Montaldo, Caterina; Giuca, Maria Rita; Iavarone, Federica; Zampino, Giuseppe; Castagnola, Massimo; Messana, Irene

    2013-07-01

    People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome.

  1. Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics*

    Science.gov (United States)

    Cabras, Tiziana; Pisano, Elisabetta; Montaldo, Caterina; Giuca, Maria Rita; Iavarone, Federica; Zampino, Giuseppe; Castagnola, Massimo; Messana, Irene

    2013-01-01

    People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10–17 yr and 18–50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization–MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome. PMID:23533003

  2. Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes

    Directory of Open Access Journals (Sweden)

    David Llères

    2017-02-01

    Full Text Available How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Förster resonance energy transfer-based fluorescence lifetime imaging microscopy (FLIM approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1 and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.

  3. Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions.

    Science.gov (United States)

    Hung, Yu-Chiang; Wang, Pei-Wen; Pan, Tai-Long

    2010-06-01

    Salvia miltiorrhiza is a Chinese herb widely used for cardiovascular disorder regimens, yet little is known about the cellular mechanisms that contribute to attenuated growth of smooth muscle cells (SMCs) under oxidative stress such as homocysteine (Hcy) treatment. As anticipated, a low dose (0.015 mg/mL) of S.miltiorrhiza aqueous extract (SMAE) significantly inhibited (>60%) the growth of a rat smooth muscle cell line (A10) under Hcy stimulation and the intracellular reactive oxygen species (ROS) concentration obviously decreased after SMAE treatment in terms of reducing p47(phox) translocation and increasing catalase activity. Signaling profile suggests that SMAE inhibited Hcy-induced A10 cell growth via the PKC/MAPK-dependent pathway. Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 14 proteins in response to Hcy and Hcy/SMAE. Meanwhile, SMAE attenuated carbonyl-modification of specific cytoskeleton and chaperone proteins leading to cell type transformation. Moreover, a network analysis using MetaCore shed more light on the molecular basis associated with SMAE efficacy. SMAE exerts its protective effect through the scavenging of ROS and subsequent modulation of protein carbonylation to inhibit cell proliferation. These signature networks and functional proteomics highlighted herein may facilitate the evaluation of potential therapeutic targets and elucidate novel mechanisms through which protein functions can be regulated by the redox status.

  4. 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.

  5. 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

    2013-02-21

    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.

  6. 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.

  7. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Directory of Open Access Journals (Sweden)

    Lorna S Kategaya

    Full Text Available Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite with external cues (e.g., daylight and food. In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2 as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

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

    Science.gov (United States)

    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.

  9. Quantitative proteomics reveal proteins enriched in tubular endoplasmic reticulum of Saccharomyces cerevisiae

    Science.gov (United States)

    Wang, Xinbo; Li, Shanshan; Wang, Haicheng; Shui, Wenqing; Hu, Junjie

    2017-01-01

    The tubular network is a critical part of the endoplasmic reticulum (ER). The network is shaped by the reticulons and REEPs/Yop1p that generate tubules by inducing high membrane curvature, and the dynamin-like GTPases atlastin and Sey1p/RHD3 that connect tubules via membrane fusion. However, the specific functions of this ER domain are not clear. Here, we isolated tubule-based microsomes from Saccharomyces cerevisiae via classical cell fractionation and detergent-free immunoprecipitation of Flag-tagged Yop1p, which specifically localizes to ER tubules. In quantitative comparisons of tubule-derived and total microsomes, we identified a total of 79 proteins that were enriched in the ER tubules, including known proteins that organize the tubular ER network. Functional categorization of the list of proteins revealed that the tubular ER network may be involved in membrane trafficking, lipid metabolism, organelle contact, and stress sensing. We propose that affinity isolation coupled with quantitative proteomics is a useful tool for investigating ER functions. DOI: http://dx.doi.org/10.7554/eLife.23816.001 PMID:28287394

  10. The proteomic signature of NPM/ALK reveals deregulation of multiple cellular pathways.

    Science.gov (United States)

    Lim, Megan S; Carlson, Mary L; Crockett, David K; Fillmore, G Chris; Abbott, David R; Elenitoba-Johnson, Olaotan F; Tripp, Sheryl R; Rassidakis, George Z; Medeiros, L Jeffrey; Szankasi, Philippe; Elenitoba-Johnson, Kojo S J

    2009-08-20

    Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.

  11. 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.

  12. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Science.gov (United States)

    Kategaya, Lorna S; Hilliard, Aisha; Zhang, Louying; Asara, John M; Ptáček, Louis J; Fu, Ying-Hui

    2012-01-01

    Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

  13. Sperm Proteomics Reveals Intensified Selection on Mouse Sperm Membrane and Acrosome Genes

    OpenAIRE

    Dorus, Steve; Wasbrough, Elizabeth R.; Busby, Jennifer; Wilkin, Elaine C.; Karr, Timothy L.

    2010-01-01

    Spermatozoa are a focal point for the impact of sexual selection due to sperm competition and sperm–female interactions in a wide range of sexually reproducing organisms. In-depth molecular investigation of the ramifications of these selective regimes has been limited due to a lack of information concerning the molecular composition of sperm. In this study, we utilize three previously published proteomic data sets in conjunction with our whole mouse sperm proteomic analysis to delineate cellu...

  14. Global Proteomics Reveal an Atypical Strategy for Carbon/Nitrogen Assimilation by a Cyanobacterium Under Diverse Environmental Perturbations*

    Science.gov (United States)

    Wegener, Kimberly M.; Singh, Abhay K.; Jacobs, Jon M.; Elvitigala, Thanura; Welsh, Eric A.; Keren, Nir; Gritsenko, Marina A.; Ghosh, Bijoy K.; Camp, David G.; Smith, Richard D.; Pakrasi, Himadri B.

    2010-01-01

    Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen. PMID:20858728

  15. Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis

    Science.gov (United States)

    Raphael, Itay; Mahesula, Swetha; Purkar, Anjali; Black, David; Catala, Alexis; Gelfond, Jonathon A. L.; Forsthuber, Thomas G.; Haskins, William E.

    2014-09-01

    Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

  16. Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

    Directory of Open Access Journals (Sweden)

    Stephens Andrew N

    2011-09-01

    Full Text Available Abstract Background Identification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW proteins. We have optimised a procedure to overcome this limitation. Results Low MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics. Conclusions This technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.

  17. “Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network”

    Energy Technology Data Exchange (ETDEWEB)

    Pertz, Olivier C.; Wang, Yingchun; Yang, Feng; Wang, Wei; gay, laurie J.; Gritsenko, Marina A.; Clauss, Therese RW; Anderson, David J.; Liu, Tao; Auberry, Kenneth J.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2008-02-12

    Neurite extension and growth cone navigation are guided by extracellular cues that control cytoskeletal rearrangements. However, understanding the complex signaling mechanisms that mediate neuritogenesis has been limited by the inability to biochemically separate the neurite and soma for spatial proteomic and bioinformatic analyses. Here, we apply global proteome profiling in combination with a novel neurite purification methodology for comparative analysis of the soma and neurite proteomes of neuroblastoma cells. The spatial relationship of 4855 proteins were mapped revealing networks of signaling proteins that control integrins, the actin cytoskeleton, and axonal guidance in the extending neurite. Bioinformatics and functional analyses revealed a spatially compartmentalized Rac/Cdc42 signaling network that operates in conjunction with multiple GEFs and GAPs to control neurite formation. Interestingly, RNA interference experiments revealed that the different GEFs and GAPs regulate specialized functions during neurite formation including neurite growth and retraction kinetics, cytoskeletal organization, and cell polarity. Our findings provide insight into the spatial organization of signaling networks that enable neuritogenesis and provide a comprehensive system-wide profile of proteins that mediate this process including those that control Rac and Cdc42 signaling.

  18. 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.

  19. Guarding against Collateral Damage during Chromatin Transactions

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Lukas, Jiri

    2013-01-01

    Signal amplifications are vital for chromatin function, yet they also bear the risk of transforming into unrestrained, self-escalating, and potentially harmful responses. Examples of inbuilt limitations are emerging, revealing how chromatin transactions are confined within physiological boundaries....

  20. Transcriptional networks and chromatin remodeling controlling adipogenesis

    DEFF Research Database (Denmark)

    Siersbæk, Rasmus; Nielsen, Ronni; Mandrup, Susanne

    2012-01-01

    remodeling have revealed 'snapshots' of this cascade and the chromatin landscape at specific time-points of differentiation. These studies demonstrate that multiple adipogenic transcription factors co-occupy hotspots characterized by an open chromatin structure and specific epigenetic modifications...

  1. 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.

  2. Proteomic analysis and functional characterization of mouse brain mitochondria during aging reveal alterations in energy metabolism.

    Science.gov (United States)

    Stauch, Kelly L; Purnell, Phillip R; Villeneuve, Lance M; Fox, Howard S

    2015-05-01

    Mitochondria are the main cellular source of reactive oxygen species and are recognized as key players in several age-associated disorders and neurodegeneration. Their dysfunction has also been linked to cellular aging. Additionally, mechanisms leading to the preservation of mitochondrial function promote longevity. In this study we investigated the proteomic and functional alterations in brain mitochondria isolated from mature (5 months old), old (12 months old), and aged (24 months old) mice as determinants of normal "healthy" aging. Here the global changes concomitant with aging in the mitochondrial proteome of mouse brain analyzed by quantitative mass-spectrometry based super-SILAC identified differentially expressed proteins involved in several metabolic pathways including glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Despite these changes, the bioenergetic function of these mitochondria was preserved. Overall, this data indicates that proteomic changes during aging may compensate for functional defects aiding in preservation of mitochondrial function. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001370 (http://proteomecentral.proteomexchange.org/dataset/PXD001370).

  3. Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria.

    Science.gov (United States)

    Hui, Sheng; Silverman, Josh M; Chen, Stephen S; Erickson, David W; Basan, Markus; Wang, Jilong; Hwa, Terence; Williamson, James R

    2015-02-12

    A central aim of cell biology was to understand the strategy of gene expression in response to the environment. Here, we study gene expression response to metabolic challenges in exponentially growing Escherichia coli using mass spectrometry. Despite enormous complexity in the details of the underlying regulatory network, we find that the proteome partitions into several coarse-grained sectors, with each sector's total mass abundance exhibiting positive or negative linear relations with the growth rate. The growth rate-dependent components of the proteome fractions comprise about half of the proteome by mass, and their mutual dependencies can be characterized by a simple flux model involving only two effective parameters. The success and apparent generality of this model arises from tight coordination between proteome partition and metabolism, suggesting a principle for resource allocation in proteome economy of the cell. This strategy of global gene regulation should serve as a basis for future studies on gene expression and constructing synthetic biological circuits. Coarse graining may be an effective approach to derive predictive phenomenological models for other 'omics' studies.

  4. Reverse genetic analysis of the yeast RSC chromatin remodeler reveals a role for RSC3 and SNF5 homolog 1 in ploidy maintenance.

    Directory of Open Access Journals (Sweden)

    Coen Campsteijn

    2007-06-01

    Full Text Available The yeast "remodels the structure of chromatin" (RSC complex is a multi-subunit "switching deficient/sucrose non-fermenting" type ATP-dependent nucleosome remodeler, with human counterparts that are well-established tumor suppressors. Using temperature-inducible degron fusions of all the essential RSC subunits, we set out to map RSC requirement as a function of the mitotic cell cycle. We found that RSC executes essential functions during G1, G2, and mitosis. Remarkably, we observed a doubling of chromosome complements when degron alleles of the RSC subunit SFH1, the yeast hSNF5 tumor suppressor ortholog, and RSC3 were combined. The requirement for simultaneous deregulation of SFH1 and RSC3 to induce these ploidy shifts was eliminated by knockout of the S-phase cyclin CLB5 and by transient depletion of replication origin licensing factor Cdc6p. Further, combination of the degron alleles of SFH1 and RSC3, with deletion alleles of each of the nine Cdc28/Cdk1-associated cyclins, revealed a strong and specific genetic interaction between the S-phase cyclin genes CLB5 and RSC3, indicating a role for Rsc3p in proper S-phase regulation. Taken together, our results implicate RSC in regulation of the G1/S-phase transition and establish a hitherto unanticipated role for RSC-mediated chromatin remodeling in ploidy maintenance.

  5. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB) Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Science.gov (United States)

    Nicolai, Serena; Filippi, Silvia; Caputo, Manuela; Cipak, Lubos; Gregan, Juraj; Ammerer, Gustav; Frontini, Mattia; Willems, Daniela; Prantera, Giorgio; Balajee, Adayabalam S; Proietti-De-Santis, Luca

    2015-01-01

    The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  6. Identification of Novel Proteins Co-Purifying with Cockayne Syndrome Group B (CSB Reveals Potential Roles for CSB in RNA Metabolism and Chromatin Dynamics.

    Directory of Open Access Journals (Sweden)

    Serena Nicolai

    Full Text Available The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER known as transcription coupled repair (TCR. CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

  7. System-wide Clinical Proteomics of Breast Cancer Reveals Global Remodeling of Tissue Homeostasis.

    Science.gov (United States)

    Pozniak, Yair; Balint-Lahat, Nora; Rudolph, Jan Daniel; Lindskog, Cecilia; Katzir, Rotem; Avivi, Camilla; Pontén, Fredrik; Ruppin, Eytan; Barshack, Iris; Geiger, Tamar

    2016-03-23

    The genomic and transcriptomic landscapes of breast cancer have been extensively studied, but the proteomes of breast tumors are far less characterized. Here, we use high-resolution, high-accuracy mass spectrometry to perform a deep analysis of luminal-type breast cancer progression using clinical breast samples from primary tumors, matched lymph node metastases, and healthy breast epithelia. We used a super-SILAC mix to quantify over 10,000 proteins with high accuracy, enabling us to identify key proteins and pathways associated with tumorigenesis and metastatic spread. We found high expression levels of proteins associated with protein synthesis and degradation in cancer tissues, accompanied by metabolic alterations that may facilitate energy production in cancer cells within their natural environment. In addition, we found proteomic differences between breast cancer stages and minor differences between primary tumors and their matched lymph node metastases. These results highlight the potential of proteomic technology in the elucidation of clinically relevant cancer signatures.

  8. Chemical proteomics with sulfonyl fluoride probes reveals selective labeling of functional tyrosines in glutathione transferases.

    Science.gov (United States)

    Gu, Christian; Shannon, D Alexander; Colby, Tom; Wang, Zheming; Shabab, Mohammed; Kumari, Selva; Villamor, Joji Grace; McLaughlin, Christopher J; Weerapana, Eranthie; Kaiser, Markus; Cravatt, Benjamin F; van der Hoorn, Renier A L

    2013-04-18

    Chemical probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chemical probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.

  9. 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.

  10. Unintended Changes in Genetically Modified Rice Expressing the Lysine-Rich Fusion Protein Gene Revealed by a Proteomics Approach

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xiang-xiang; TANG Tang; LIU Fu-xia; LU Chang-li; HU Xiao-lan; JI Li-lian; LIU Qiao-quan

    2013-01-01

    Development of new technologies for evaluating genetically modiifed (GM) crops has revealed that there are unintended insertions and expression changes in GM crops. Proifling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops. Here, we report the application of a comparative proteomic approach to investigate the protein proifle differences between a GM rice line, which has a lysine-rich protein gene, and its non-transgenic parental line. Proteome analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrum analysis of the seeds identiifed 22 differentially expressed protein spots. Apart from a number of glutelins that were detected as targeted proteins in the GM line, the majority of the other changed proteins were involved in carbohydrate metabolism, protein synthesis and stress responses. These results indicated that the altered proteins were not associated with plant allergens or toxicity.

  11. Comparative Proteome Analysis of the Tuberous Roots of Six Cassava (Manihot esculenta) Varieties Reveals Proteins Related to Phenotypic Traits.

    Science.gov (United States)

    Schmitz, Gabriela Justamante Händel; de Magalhães Andrade, Jonathan; Valle, Teresa Losada; Labate, Carlos Alberto; do Nascimento, João Roberto Oliveira

    2016-04-27

    Cassava (Manihot esculenta Crantz) is a staple food and an important source of starch, and the attributes of its tuberous root largely depend on the variety. The proteome of cassava has been investigated; however, to date, no study has focused on varieties that reveal the molecular basis of phenotypical characteristics. Therefore, we aimed to compare the proteome of the tuberous roots of six cassava varieties that differed in carbohydrates, carotenoids, and resistance to diseases, among other attributes. Two-dimensional gels showed 146 differential spots between the varieties, and the functional roles of some differential proteins were correlated to phenotypic characteristics of the varieties, such as the amount of carbohydrates or carotenoids and the resistance to biotic or abiotic stresses. The results obtained here highlight elements that might help to direct the improvement of new cultivars of cassava, which is an economically and socially relevant crop worldwide.

  12. Global Proteomics Reveal An Atypical Strategy for Carbon/Nitrogen Assimilation by a Cyanobacterium Under Diverse Environmental Perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Wegener, Kimberly M.; Singh, Abhay K.; Jacobs, Jon M.; Elvitigala, Thanura R.; Welsh, Eric A.; Keren, Nir S.; Gritsenko, Marina A.; Ghosh, Bijoy K.; Camp, David G.; Smith, Richard D.; Pakrasi, Himadri B.

    2010-12-01

    Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a largescale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1,955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1,198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen. Oxygenic phototrophic prokaryotes, the progenitors of the chloroplast, are crucial to global oxygen production and worldwide carbon and nitrogen cycles. These microalgae are robust organisms capable carbon neutral biofuel production. Synechocystis sp. PCC 6803 has historically been a model cyanobacterium for photosynthetic research and is emerging as a promising biofuel platform. Cellular responses are severely modified by environmental

  13. Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

    Science.gov (United States)

    Wang, Jinhui; Valo, Zuzana; Bowers, Chauncey W; Smith, David D; Liu, Zheng; Singer-Sam, Judith

    2010-11-04

    As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay). We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1) hybrid clonal neural stem cell (NSC) lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

  14. Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

    Directory of Open Access Journals (Sweden)

    Jinhui Wang

    Full Text Available As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay. We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1 hybrid clonal neural stem cell (NSC lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

  15. AT1 receptor induced alterations in histone H2A reveal novel insights into GPCR control of chromatin remodeling.

    Directory of Open Access Journals (Sweden)

    Rajaganapathi Jagannathan

    Full Text Available Chronic activation of angiotensin II (AngII type 1 receptor (AT(1R, a prototypical G protein-coupled receptor (GPCR induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT(1R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT(1R modify the composition of histone isoforms and post-translational modifications (PTM, thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT(1R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT(1R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT(1R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.

  16. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

    DEFF Research Database (Denmark)

    Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A

    2012-01-01

    The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and ...

  17. 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.

  18. 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.

  19. Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules

    NARCIS (Netherlands)

    Rajan, B.; Fernandes, J.M.O.; Caipang, C.M.A.; Kiron, V.; Rombout, J.H.W.M.; Brinchmann, M.

    2011-01-01

    The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC–MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontol

  20. 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-s...

  1. Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion

    NARCIS (Netherlands)

    Ellen, Albert F.; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2010-01-01

    Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles

  2. Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.

    NARCIS (Netherlands)

    Khan, S.; Franke-Fayard, B.; Mair, G.R.; Lasonder, E.; Janse, C.J.; Mann, M.; Waters, A.P.

    2005-01-01

    Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic pa

  3. 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).

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

    NARCIS (Netherlands)

    Swelm, R.P.L. van; Laarakkers, C.M.; Pertijs, J.C.L.M.; Verweij, V.G.M.; Masereeuw, R.; Russel, F.G.M.

    2013-01-01

    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

  5. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum-Phytophthora capsici Phytopathosystem.

    Science.gov (United States)

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  6. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum—Phytophthora capsici Phytopathosystem

    Science.gov (United States)

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  7. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

    Science.gov (United States)

    Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

    2016-08-01

    The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  8. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin.

  9. Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics.

    Science.gov (United States)

    Barathi, Veluchamy A; Chaurasia, Shyam S; Poidinger, Michael; Koh, Siew Kwan; Tian, Dechao; Ho, Candice; Iuvone, P Michael; Beuerman, Roger W; Zhou, Lei

    2014-11-07

    Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (-15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

  10. Comparative proteomic and physiological analyses reveal the protective effect of exogenous calcium on the germinating soybean response to salt stress.

    Science.gov (United States)

    Yin, Yongqi; Yang, Runqiang; Han, Yongbin; Gu, Zhenxin

    2015-01-15

    suppressed under salt stress condition. According to previous studies, exogenous calcium counters the harmful effect of salt stress and increases the biomass and GABA content of germinating soybeans. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unknown. This paper is the first study employing comparative proteomic and physiological analyses to reveal the protective effect of exogenous calcium in the germinating soybean response to salt stress. Our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. The functions of those significantly changed proteins are also analyzed. The physiological and comparative proteomic analyses revealed the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. The findings from this paper are beneficial to high GABA-rich germinating soybean biomass. Additionally, these findings also might be applicable to the genetic engineering of soybean plants to improve stress tolerance.

  11. Pathobiochemical Changes in Diabetic Skeletal Muscle as Revealed by Mass-Spectrometry-Based Proteomics

    Directory of Open Access Journals (Sweden)

    Kay Ohlendieck

    2012-01-01

    Full Text Available Insulin resistance in skeletal muscle tissues and diabetes-related muscle weakness are serious pathophysiological problems of increasing medical importance. In order to determine global changes in the protein complement of contractile tissues due to diabetes mellitus, mass-spectrometry-based proteomics has been applied to the investigation of diabetic muscle. This review summarizes the findings from recent proteomic surveys of muscle preparations from patients and established animal models of type 2 diabetes. The potential impact of novel biomarkers of diabetes, such as metabolic enzymes and molecular chaperones, is critically examined. Disease-specific signature molecules may be useful for increasing our understanding of the molecular and cellular mechanisms of insulin resistance and possibly identify new therapeutic options that counteract diabetic abnormalities in peripheral organ systems. Importantly, the biomedical establishment of biomarkers promises to accelerate the development of improved diagnostic procedures for characterizing individual stages of diabetic disease progression, including the early detection of prediabetic complications.

  12. Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits

    OpenAIRE

    Liu, Xialin; Min ZHANG; Liu, Yuhua; Challa, Pratap; Gonzalez, Pedro; Liu, Yizhi

    2008-01-01

    Purpose To explore lens crystallin characteristics and morphology of rabbit regenerated lenses in comparison with wild type natural lenses by means of proteomic analysis and histological assay. Methods The lens regeneration model of the New Zealand rabbit was established, and lens regeneration was observed by slit lamp examination and photography. A histological assay was evaluated under light microscopy and transmission electron microscopy (TEM). Protein samples of regenerated lenses were co...

  13. Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

    Science.gov (United States)

    2009-08-27

    possible to develop proteomics assays that are sensitive enough to allow presymptomatic diagnosis . TA B LE II S im ila rit es am on g cr os s- re ac tiv...potential acts of terrorism. Because human deaths may occur within 48 h of infection (7), delays in proper diagnosis have led to disease complications...infectious agent by amplification of genetic markers. Although host an- tibody responses provide a sensitive indicator of current or past infection

  14. Proteomic and Functional Analyses Reveal MAPK1 Regulates Milk Protein Synthesis

    OpenAIRE

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

    2012-01-01

    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 mamm...

  15. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    Science.gov (United States)

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis.

  16. 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.

  17. Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction.

    Science.gov (United States)

    Bakun, M; Senatorski, G; Rubel, T; Lukasik, A; Zielenkiewicz, P; Dadlez, M; Paczek, L

    2014-02-01

    Aging is a complex physiological process that poses considerable conundrums to rapidly aging societies. For example, the risk of dying from cardiovascular diseases and/or cancer steadily declines for people after their 60s, and other causes of death predominate for seniors older than 80 years of age. Thus, physiological aging presents numerous unanswered questions, particularly with regard to changing metabolic patterns. Urine proteomics analysis is becoming a non-invasive and reproducible diagnostic method. We investigated the urine proteomes in healthy elderly people to determine which metabolic processes were weakened or strengthened in aging humans. Urine samples from 37 healthy volunteers aged 19-90 years (19 men, 18 women) were analyzed for protein expression by liquid chromatography-tandem mass spectrometry. This generated a list of 19 proteins that were differentially expressed in different age groups (young, intermediate, and old age). In particular, the oldest group showed protein changes reflective of altered extracellular matrix turnover and declining immune function, in which changes corresponded to reported changes in cardiovascular tissue remodeling and immune disorders in the elderly. Thus, urinary proteome changes in the elderly appear to reflect the physiological processes of aging and are particularly clearly represented in the circulatory and immune systems. Detailed identification of "protein trails" creates a more global picture of metabolic changes that occur in the elderly.

  18. 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

  19. Phloem Proteomics Reveals New Aspects of Long-distance Signaling in Plants

    Institute of Scientific and Technical Information of China (English)

    Urs F.Benning; Banita Tamot; Susanne Hoffmann-Benning

    2012-01-01

    As the world population grows our need for food and fuel increases drastically.Given the additional impact of the global climate change a second "green revolution",however seems unlikely to be achieved by simply adding fertilizer or accessing new land but will need a better understanding of the factors and processes essential for plant growth and development.One of those processes is the adaptation of plants to their environment.Since plants are sessile and cannot move to appropriate hiding places or feeding grounds to escape adverse conditions,they evolved mechanisms to detect changes in their environment,communicate these to different organs,and adjust development accordingly.One of these adaptations,the phloem,serves as a major trafficking pathway for assimilates,viruses,RNA,plant hormones,metabolites,and proteins with functions ranging from synthesis to metabolism to signaling.The study of signaling compounds within the phloem is essential for our understanding of plant communication of environmental cues.Determining the nature of signals and the mechanisms by which they are communicated through the phloem will lead to a more complete understanding of plant development and plant responses to stress and,as a result could lead to the development of plants with increased adaptation to a changed environment.Our analysis of Arabidopsis phloem exudates revealed several lipid-binding proteins as well as lipids.Lipids are not typically expected in the aqueous environment of SEs.Yet the long-distance transport of hydrophobic compounds in aqueous systems is not without precedence in biological systems:In human blood lipids are often bound to proteins and transported for storage,use,modification,or degradation; alternatively,they serve as messengers and modulate transcription factor activity and,as a result,development.It is conceivable that lipids and the respective lipid-binding proteins in the phloem serve similar functions in plants and play an important role in

  20. Physiological and proteomic analyses of leaves from the halophyte Tangut Nitraria reveals diverse response pathways critical for high salinity tolerance

    Directory of Open Access Journals (Sweden)

    Tielong eCheng

    2015-02-01

    Full Text Available Soil salinization poses a serious threat to the environment and agricultural productivity worldwide. Studies on the physiological and molecular mechanisms of salinity tolerance in halophytic plants provide valuable information to enhance their salt tolerance. Tangut Nitraria is a widely distributed halophyte in saline–alkali soil in the northern areas of China. In this study, we used a proteomic approach to investigate the molecular pathways of the high salt tolerance of T. Nitraria. We analyzed the changes in biomass, photosynthesis, and redox-related enzyme activities in T. Nitraria leaves from plant seedlings treated with high salt concentration. Comparative proteomic analysis of the leaves revealed that the expression of 71 proteins was significantly altered after salinity treatments of T. Nitraria. These salinity-responsive proteins were mainly involved in photosynthesis, redox homeostasis, stress/defense, carbohydrate and energy metabolism, protein metabolism, signal transduction, and membrane transport. Results showed that the reduction of photosynthesis under salt stress was attributed to the down-regulation of the enzymes and proteins involved in the light reaction and Calvin cycle. Protein–protein interaction analysis revealed that the proteins involved in redox homeostasis, photosynthesis, and energy metabolism constructed two types of response networks to high salt stress. T. Nitraria plants developed diverse mechanisms for scavenging reactive oxygen species in their leaves to cope with stress induced by high salinity. This study provides important information regarding the salt tolerance of the halophyte T. Nitraria.

  1. Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males.

    Science.gov (United States)

    Zhang, Sheng; Zhang, Yunxiang; Cao, Yanchun; Lei, Yanbao; Jiang, Hao

    2016-03-04

    Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency.

  2. The spider hemolymph clot proteome reveals high concentrations of hemocyanin and von Willebrand factor-like proteins.

    Science.gov (United States)

    Sanggaard, Kristian W; Dyrlund, Thomas F; Bechsgaard, Jesper S; Scavenius, Carsten; Wang, Tobias; Bilde, Trine; Enghild, Jan J

    2016-02-01

    Arthropods include chelicerates, crustaceans, and insects that all have open circulation systems and thus require different properties of their coagulation system than vertebrates. Although the clotting reaction in the chelicerate horseshoe crab (Family: Limulidae) has been described in details, the overall protein composition of the resulting clot has not been analyzed for any of the chelicerates. The largest class among the chelicerates is the arachnids, which includes spiders, ticks, mites, and scorpions. Here, we use a mass spectrometry-based approach to characterize the spider hemolymph clot proteome from the Brazilian whiteknee tarantula, Acanthoscurria geniculata. We focused on the insoluble part of the clot and demonstrated high concentrations of proteins homologous to the hemostasis-related and multimerization-prone von Willebrand factor. These proteins, which include hemolectins and vitellogenin homologous, were previously identified as essential components of the hemolymph clot in crustaceans and insects. Their presence in the spider hemolymph clot suggests that the origin of these proteins' function in coagulation predates the split between chelicerates and mandibulata. The clot proteome reveals that the major proteinaceous component is the oxygen-transporting and phenoloxidase-displaying abundant hemolymph protein hemocyanin, suggesting that this protein also plays a role in clot biology. Furthermore, quantification of the peptidome after coagulation revealed the simultaneous activation of both the innate immune system and the coagulation system. In general, many of the identified clot-proteins are related to the innate immune system, and our results support the previously suggested crosstalk between immunity and coagulation in arthropods.

  3. Trophic Mode-Dependent Proteomic Analysis Reveals Functional Significance of Light-Independent Chlorophyll Synthesis in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Fang, Longfa; Ge, Haitao; Huang, Xiahe; Liu, Ye; Lu, Min; Wang, Jinlong; Chen, Weiyang; Xu, Wu; Wang, Yingchun

    2017-01-09

    The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein profile changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotrophic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of PII phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study using a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth.

  4. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology.

    Science.gov (United States)

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-02-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

  5. Proteome profiling reveals regional protein alteration in cerebrum of common marmoset (Callithrix jacchus) exposed to methylmercury.

    Science.gov (United States)

    Shao, Yueting; Yamamoto, Megumi; Figeys, Daniel; Ning, Zhibin; Chan, Hing Man

    2016-03-10

    Methylmercury (MeHg) is known to selectively damage the calcarine and precentral cortices along deep sulci and fissures in adult cases, but the detailed mechanism is still unclear. This study aims to identify and analyze the differential proteome expression in two regions of the cerebrum (the frontal lobe and the occipital lobe including the calcarine sulcus) of the common marmoset exposed to MeHg using a shot-gun proteomic approach. A total of 1045 and 1062 proteins were identified in the frontal lobe (FL) and occipital lobe (OL), of which, 62 and 89 proteins were found significantly changed with MeHg exposure. Functional enrichment/depletion analysis showed that the lipid metabolic process and proteolysis were affected in both two lobes. Functional changes in FL were characterized in cell cycle and cell division, sulfur compound metabolic process, microtubule-based process and glycerolipid metabolic process. In comparison, proteins were enriched in the functions of transport, carbohydrate metabolic process, chemical caused homeostasis and regulation of body fluid levels in OL. Pathway analysis predicted that vasopressin-regulated water reabsorption was disturbed in MeHg-treated FL. Our results showed that MeHg induced regional specific protein changes in FL and OL but with similar endpoint effects such as energy diminish and disruption of water transport. APOE and GPX1 were shown to be possible key proteins targeted by MeHg leading to multiple functional changes in OL. This is the first report of the whole proteome changes of primate cerebrum for MeHg neurotoxicity, and the results will contribute to the understanding of molecular basis of MeHg intoxication in humans.

  6. Initial Morphogenetic Features of Proteome of Suprastructures of Interphase Chromatin for Germination of Mature Germs in Conditions of Adapting to Winter in Wheat

    Directory of Open Access Journals (Sweden)

    Ivanova E.A.

    2015-12-01

    Full Text Available Investigation morphogenetic processes of how the plant is formed for interact with the environment is necessary to understanding the fundamental problems of stress supramolecular biochemistry of developmental biology. Modern biology has established that the molecular morphogenesis belonging to the number of morphological processes. The molecular morphogenesis is directly based on well-studied forces of intermolecular interactions. From the progress of understanding the morphogenesis, expected not only purely cognitive, but also the practical results of biotechnology. The purpose of work was the analysis of shielding of morphogenetic suprastructures of interphase chromatin matrix to the localization of sensitivity of the regulatory proteolysis in the conditions of adaptation of vegetative phase of wheat to the stressful environmental factors. It was shown that in the conditions of long-term adaptation to cold shock of wheat at the level of suprastructures, of the packaged in the nuclear matrix of the genome, is carried out the total shielding of arginine-X zones for the regulatory protease-processing. Perhaps these zones represents one of the elements of the LCR (locus control regions, which are related to the histone code and influence the architectural organization of the cell nucleus, which is formed to facilitate life of organism in complex environmental conditions. These data will be useful for those who involved in the development of mathematical-logic schemes of theory and practice of biological specificity and may be included in the ontology of stages of plant growth and development.

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

    DEFF Research Database (Denmark)

    Yang, Fen; Melo-Braga, Marcella N; Larsen, Martin Røssel;

    2013-01-01

    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......-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...

  8. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins

    DEFF Research Database (Denmark)

    Cappellini, E.; Jensen, L.J.; Szklarczyk, D.;

    2012-01-01

    We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low...... evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth (Mammuthus columbi) samples from temperate latitudes, extending the potential of the approach...

  9. Proteomic Stable Isotope Probing Reveals Taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton.

    Science.gov (United States)

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; Hettich, Robert L; Mayali, Xavier; Pan, Chongle; Mueller, Ryan S

    2016-01-01

    Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual primary production, yet defining how substrate utilization preferences and resource partitioning structure microbial communities remains a challenge. In this study, proteomic stable isotope probing (proteomic SIP) was used to characterize population-specific assimilation of dissolved free amino acids (DFAAs), a major source of dissolved organic carbon for bacterial secondary production in aquatic environments. Microcosms of seawater collected from Newport, Oregon, and Monterey Bay, California, were incubated with 1 µM (13)C-labeled amino acids for 15 and 32 h. The taxonomic compositions of microcosm metaproteomes were highly similar to those of the sampled natural communities, with Rhodobacteriales, SAR11, and Flavobacteriales representing the dominant taxa. Analysis of (13)C incorporation into protein biomass allowed for quantification of the isotopic enrichment of identified proteins and subsequent determination of differential amino acid assimilation patterns between specific bacterioplankton populations. Proteins associated with Rhodobacterales tended to have a significantly high frequency of (13)C-enriched peptides, opposite the trend for Flavobacteriales and SAR11 proteins. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of (13)C-enriched spectra at time point 2. Alteromonadales proteins also had a significantly high frequency of (13)C-enriched peptides, particularly within ribosomal proteins, demonstrating their rapid growth during incubations. Overall, proteomic SIP facilitated quantitative comparisons of DFAA assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population level metabolic responses to resource acquisition in complex microbial communities

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. [Heat-responsive mechanisms in plants revealed by proteomic analysis: A review].

    Science.gov (United States)

    Liu, Jun-ming; Zhao, Qi; Yin, Ze-peng; Xu, Chen-xi; Wang, Quan-hua; Dai, Shao-jun

    2015-08-01

    Heat stress is a major abiotic stress that limits plant growth and productivity. In recent years, proteomic investigations provide more information for understanding the sophisticated heat-responsive molecular mechanism in plants at systematic biological level. The heat-responsive proteomic patterns in several plants, i. e., model plants (Arabidopsis thaliana), staple food crops (soybean, rice and wheat), heat-tolerant plants (Agrostis stolonifera, Portulaca oleracea, and Carissa spinarum), grapevine, Populus euphratica, Medicago sativa, and Pinellia ternate, were reported. A total of 838 heat-responsive proteins have been identified in these studies. Among them, 534 proteins were induced and the expression of 304 proteins was reduced in plants under heat stress. In this paper, the diverse protein patterns in plants under various heat stress conditions (30-45 °C for 0-10 d) were analyzed integratively. This provided new evidences and clues for further interpreting the signaling and metabolic pathways, e.g., signaling, stress and defense, carbohydrate and energy metabolism, photosynthesis, transcription, protein synthesis and fate, membrane and transport, in heat-responsive networks, and laid a foundation for a holistic understanding of the molecular regulatory mechanism in plants in response to heat stress.

  13. Medicago truncatula root nodule proteome analysis reveals differential plant and bacteroid responses to drought stress.

    Science.gov (United States)

    Larrainzar, Estíbaliz; Wienkoop, Stefanie; Weckwerth, Wolfram; Ladrera, Rubén; Arrese-Igor, Cesar; González, Esther M

    2007-07-01

    Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula 'Jemalong A17' plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

  14. Proteome analysis of tobacco leaves reveals dynamic changes in protein expression among different cultivation areas.

    Science.gov (United States)

    Lu, L M; Ye, K Y; Tang, Z X; Liu, L; Chen, Y; Luo, J; Huang, Y B

    2015-11-30

    The leaves of tobacco plants were used to analyze differences in protein content of tobacco grown in the four main flue-cured tobacco-producing areas of Sichuan Province, China. An improved protein extraction method, isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gel electrophoretic separation, was used to extract and separate total protein from tobacco leaves. Proteomic maps with relatively high resolution and repeatability were produced. At isoelectric points 4 to 7 and molecular weight ranging from 20-100 kDa, we detected 1032, 1030, 1019, and 1011 clearly visible protein spots in tobacco leaves from the four study areas. Proteome comparison between these protein spots showed that 119 spots with a greater than 2-fold change in expression quantity contributed to the variation in expression. Of which, 115 were successfully identified and annotated. According to the annotation results, these proteins participate in photosynthesis, energy metabolism, mineral nutrition, terpene metabolism, defensive reaction, and other physiological and biochemical processes. This study preliminarily explains the effects of ecological conditions on the physiological metabolism of tobacco leaves and how such effects directly or indirectly contribute to tobacco leaf quality.

  15. Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation

    Science.gov (United States)

    Graf, Alexander; Coman, Diana; Walsh, Sean; Flis, Anna; Stitt, Mark; Gruissem, Wilhelm

    2017-01-01

    The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes. PMID:28250106

  16. Proteomic analysis reveals key proteins and phosphoproteins upon seed germination of wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Kun eDong

    2015-11-01

    Full Text Available Wheat (Triticum aestivum L. is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE-based proteomic approach. A total of 166 differentially expressed protein (DEP spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/ transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

  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. 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.

  19. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

    Science.gov (United States)

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong

    2015-11-20

    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  20. Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

    Science.gov (United States)

    Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

    2016-01-01

    Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

  1. 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.

  2. Integration of Transcriptome and Proteome Reveals the Alkaloids Biosynthesis in Macleaya cordata and Macleaya microcarpa

    Institute of Scientific and Technical Information of China (English)

    Yisong Liu; Wei Liu; Xiubing Liu; Peng Huang; Pengcheng Zhu; Pi Cheng; Jing Zeng

    2012-01-01

    The Macleaya spp.,including Macleaya cordata and Macleaya microcarpa,are traditional anti-virus,inflammation eliminating,and insecticide herb medicines for their isoquinoline alkaloids.The studies of their alkaloids biosyntheses are urgent for better application.To further characterize their alkaloids biosyntheses,we elaborately designed the transcriptome,proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosyntheses.From the transcriptome data,we obtained 69367 and 78255 unigenes for M.cordata and M.microcarpa,which two thirds of them were similar to sequences in public databases.By metabolism profiling,we observed reverse patterns in different organs of two species for alkaloids sanguinarine,chelerythrine,protopine,and allocryptopine.Thus,the expression of enzymes in alkaloid biosynthesis pathways and the differential gene expression for multiple interesting comparisons were analyzed.We identified more than 1000 proteins and hundreds of differentially expressed proteins from iTRAQ proteome data.Furthermore,the ultrastructure of laticifers by SEM proved the alkaloids accumulation in the mature roots.This study suggests strongly that root maybe the organ for major alkaloids biosynthesis.Except for biosynthesis,the alkaloids storage and transport were also important for their accumulation.This work provided the first genome scale analysis for Macleaya spp.and shed light on researches for non-model plants by integrating different high-throughput technologies.

  3. 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

    2015-11-01

    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.

  4. 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.

  5. Dynamic proteomic analysis reveals a switch between central carbon metabolism and alcoholic fermentation in rice filling grains.

    Science.gov (United States)

    Xu, Sheng Bao; Li, Tang; Deng, Zhu Yun; Chong, Kang; Xue, Yongbiao; Wang, Tai

    2008-10-01

    Accumulation of reserve materials in filling grains involves the coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice (Oryza sativa) is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 d after flowering (DAF); thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout eight sequential developmental stages from 6 to 20 DAF and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation, whereas the down-regulated proteins in the process were involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis, and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.

  6. 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.

  7. 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.

  8. In Depth Proteome Analysis of Ripening Muscadine Grape Berry cv. Carlos Reveals Proteins Associated with Flavor and Aroma Compounds.

    Science.gov (United States)

    Kambiranda, Devaiah; Basha, Sheikh M; Singh, Rakesh K; He, Huan; Calvin, Kate; Mercer, Roger

    2016-09-02

    Ripening in nonclimacteric fruits such as grape involves complex chemical changes that have a profound influence on the accumulation of flavor and aroma compounds distinct to a particular grape genotype. In this study, proteome characterization of wine type bronze muscadine grape (Vitis rotundifolia cv. Carlos), primarily grown in the Southeastern United States was performed during berry ripening. Stage-specific protein expression was obtained among different stages of berries. Differential analysis showed the expression of 522 proteins that regulate diverse biological processes and metabolic pathways. Of these, 30 proteins are associated with the production of key phenolic compounds, whereas 25 are associated with the production of muscadine aroma compounds. These proteins are involved in the phenylpropanoid pathway, terpene synthesis, fatty acid derived volatiles and esters that affect muscadine berry flavor and aroma characteristics. Further, gene expression analysis during ripening validated the expression pattern of 12 proteins. Catechin, epicatechin, and four stilbenes were quantified to correlate observed proteome changes. This study not only revealed biochemical changes during muscadine berry ripening but also offers indicators for marker-assisted breeding to enhance organoleptic properties of muscadine grape to improve its flavor and aroma properties.

  9. Extreme diversity of scorpion venom peptides and proteins revealed by transcriptomic analysis: implication for proteome evolution of scorpion venom arsenal.

    Science.gov (United States)

    Ma, Yibao; He, Yawen; Zhao, Ruiming; Wu, Yingliang; Li, Wenxin; Cao, Zhijian

    2012-02-16

    Venom is an important genetic development crucial to the survival of scorpions for over 400 million years. We studied the evolution of the scorpion venom arsenal by means of comparative transcriptome analysis of venom glands and phylogenetic analysis of shared types of venom peptides and proteins between buthids and euscorpiids. Fifteen types of venom peptides and proteins were sequenced during the venom gland transcriptome analyses of two Buthidae species (Lychas mucronatus and Isometrus maculatus) and one Euscorpiidae species (Scorpiops margerisonae). Great diversity has been observed in translated amino acid sequences of these transcripts for venom peptides and proteins. Seven types of venom peptides and proteins were shared between buthids and euscorpiids. Molecular phylogenetic analysis revealed that at least five of the seven common types of venom peptides and proteins were likely recruited into the scorpion venom proteome before the lineage split between Buthidae and Euscorpiidae with their corresponding genes undergoing individual or multiple gene duplication events. These are α-KTxs, βKSPNs (β-KTxs and scorpines), anionic peptides, La1-like peptides, and SPSVs (serine proteases from scorpion venom). Multiple types of venom peptides and proteins were demonstrated to be continuously recruited into the venom proteome during the evolution process of individual scorpion lineages. Our results provide an insight into the recruitment pattern of the scorpion venom arsenal for the first time.

  10. 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

  11. Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2016-08-01

    Full Text Available The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS and identified nucleolus-associated chromatin domains (NADs by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1. NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs, sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

  12. Comparative proteomics of milk fat globule membrane in different species reveals variations in lactation and nutrition.

    Science.gov (United States)

    Lu, Jing; Wang, Xinyu; Zhang, Weiqing; Liu, Lu; Pang, Xiaoyang; Zhang, Shuwen; Lv, Jiaping

    2016-04-01

    In present study, 312, 554, 175 and 143 proteins were identified and quantified by label-free quantitative proteomics in human, cow, goat and yak milk fat globule membrane (MFGM), respectively. Fifty proteins involved in vesicle mediate transport and milk fat globule secretion were conserved among species. Moreover, proteins involved in lipid synthesis and secretion (xanthine dehydrogenase/oxidase, stomatin and CD36), showed different expression pattern and the host defense proteins exhibited various profiles within species. Notably, the content and activity of lipid catabolic enzymes were significantly higher in human MFGM, which could be indicative of the superior fat utilization in breast fed infants. Our findings unraveled the significant differences in protein composition of human milk and conventionally used substitutes of it. The in-depth study of lipid metabolic enzymes in human MFGM will probably contribute to the improvement of the fat utilization through modulation of lipid catabolic enzymes in infant formula.

  13. Comparative proteomic analysis reveals mite (Varroa destructor) resistance-related proteins in Eastern honeybees (Apis cerana).

    Science.gov (United States)

    Ji, T; Shen, F; Liu, Z; Yin, L; Shen, J; Liang, Q; Luo, Y X

    2015-08-21

    The mite (Varroa destructor) has become the greatest threat to apiculture worldwide. As the original host of the mite, Apis cerana can effectively resist the mite. An increased understanding of the resistance mechanisms of Eastern honeybees against V. destructor may help researchers to protect other species against these parasites. In this study, the proteomes of 4 Apis cerana colonies were analyzed using an isobaric tag for relative and absolute quantitation technology. We determined the differences in gene and protein expression between susceptible and resistant colonies that were either unchallenged or challenged by V. destructor. The results showed that a total of 1532 proteins were identified. Gene Ontology enrichment analysis suggested that the transcription factors and basic metabolic and respiratory processes were efficient and feasible factors controlling this resistance, and 12 differentially expressed proteins were identified in Venn analysis. The results were validated by quantitative polymerase chain reaction. This study may provide insight into the genetic mechanisms underlying the resistance of honeybee to mites.

  14. Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion.

    Science.gov (United States)

    Ellen, Albert F; Albers, Sonja-Verena; Driessen, Arnold J M

    2010-01-01

    Although a large number of potentially secreted proteins can be predicted on the basis of genomic distribution of signal sequence-bearing proteins, protein secretion in Archaea has barely been studied. A proteomic inventory and comparison of the growth medium proteins in three hyperthermoacidophiles, i.e., Sulfolobus solfataricus, S. acidocaldarius and S. tokodaii, indicates that only few proteins are freely secreted into the growth medium and that the majority originates from cell envelope bound forms. In S. acidocaldarius both cell-associated and secreted alpha-amylase activities are detected. Inactivation of the amyA gene resulted in a complete loss of activity, suggesting that the same protein is responsible for the a-amylase activity at both locations. It is concluded that protein secretion in Sulfolobus is a limited process, and it is suggested that the S-layer may act as a barrier for the free diffusion of folded proteins into the medium.

  15. 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

    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...... 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...... directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance...

  16. iTRAQ quantitative proteomic analysis reveals the pathways for methanation of propionate facilitated by magnetite

    DEFF Research Database (Denmark)

    Jing, Yuhang; Wan, Jingjing; Angelidaki, Irini

    2017-01-01

    by around 44% in batch experiments, and both direct interspecies electron transfer and interspecies H2 transfer were thermodynamically feasible with the addition of magnetite. The methanation of propionate facilitated by magnetite was also demonstrated in a long-term operated continuous reactor. The methane...... enriched with the addition of magnetite. iTRAQ quantitative proteomic analysis, which was used in mixed culture for the first time, showed that magnetite induced the changes of protein expression levels involved in various pathways during the methanation of propionate. The up-regulation of proteins...... electron transfer considering its up-regulation with the addition of magnetite and origination from Thauera. Most of the up-regulated proteins in methane metabolism were originated from Methanosaeta, while most of the enzymes with down-regulated proteins were originated from Methanosarcina. However, the up-regulated...

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Comparative proteomics reveals a significant bias toward alternative protein isoforms with conserved structure and function.

    Science.gov (United States)

    Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L

    2012-09-01

    Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of "novel" and "putative" protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and

  19. Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation.

    Science.gov (United States)

    Tahir, Ammar; Bileck, Andrea; Muqaku, Besnik; Niederstaetter, Laura; Kreutz, Dominique; Mayer, Rupert L; Wolrab, Denise; Meier, Samuel M; Slany, Astrid; Gerner, Christopher

    2017-02-07

    During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

  20. A Polymer Model with Epigenetic Recolouring Reveals a Pathway for the de novo Establishment and 3D Organisation of Chromatin Domains

    CERN Document Server

    Michieletto, Davide; Marenduzzo, Davide

    2016-01-01

    One of the most important problems in development is how epigenetic domains can be first established, and then maintained, within cells. To address this question, we propose a framework which couples 3D chromatin folding dynamics, to a "recolouring" process modelling the writing of epigenetic marks. Because many intra-chromatin interactions are mediated by bridging proteins, we consider a "two-state" model with self-attractive interactions between two epigenetic marks which are alike (either active or inactive). This model displays a first-order-like transition between a swollen, epigenetically disordered, phase, and a compact, epigenetically coherent, chromatin globule. If the self-attraction strength exceeds a threshold, the chromatin dynamics becomes glassy, and the corresponding interaction network freezes. By modifying the epigenetic read-write process according to more biologically-inspired assumptions, our polymer model with recolouring recapitulates the ultrasensitive response of epigenetic switches t...

  1. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    OpenAIRE

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been u...

  2. Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres

    Directory of Open Access Journals (Sweden)

    Laura Garcia-Exposito

    2016-11-01

    Full Text Available Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT pathways for telomere maintenance and survival. ALT involves homologous recombination (HR-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID, we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη. We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

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

    Directory of Open Access Journals (Sweden)

    Patrick C Y Woo

    2009-03-01

    Full Text Available 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

  4. Wheat Mitochondrial Proteomes Reveal Links between Mitochondrial Respiration, Antioxidant Defence and Plant Salinity Tolerance

    Institute of Scientific and Technical Information of China (English)

    Richard P.Jacoby; A.Harvey Millar; Nicolas L.Taylor

    2012-01-01

    Mitochondrial respiration extracts chemical energy from carbon-containing molecules,and converts that energy into ATP,the cellular energy currency.The ATP produced by respiration fuels biochemical and physiological processes that enable the plant to survive and grow.Several studies have observed a negative correlation between respiration rate and growth rate,indicating that respiratory properties might influence biomass accumulation.Furthermore,there is evidence that salinity-sensitive wheat varieties display a higher respiration rate under salt treatment,while salt-tolerant varieties maintains similar a respiration rate under both control and salt treatments.However,the molecular basis of such results remains unexplored.Here we have investigated the mitochondrial proteome and differences associated with salt tolerance in two Australian commercial varieties of wheat.Using 2D-DIGE we have found quantitative differences in the shoot mitochondrial proteomes of Triticum aestivum v.Wyalkatchem and v.Janz,two commercially important wheat varieties that are known from a range of experiments to have differing salinity tolerance.These proteins included Mn-superoxide dismutase (Mn-SOD),cysteine synthase,nucleotide diphosphate kinase and the voltage dependent anion channel (VDAC).Antibodies to the mitochondrial alternative oxidase (AOX),previously linked to reduced reactive oxygen species (ROS) formation from the electron transport chain and salt tolerance in Arabidopsis,also showed a commensurate higher abundance in v.Wyakatchem in both control and salt-treated conditions.To further investigate this intial observation we screened 24 west australian wheat varieties for biomass retention when subjected to salt stress in a hydroponic system in a growth cabinet,with v.Krichauff and v.Westonia being the top performers.In addition we have investigated the the biomass and respiration rates in a subset of these varieties grown in a salt-affected field in the WA wheatbelt

  5. Data on the kinetics of in vitro assembled chromatin.

    Science.gov (United States)

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Schmidt, Andreas; Imhof, Axel

    2016-09-01

    Here, we use LC-MS/MS and SWATH-MS to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos (DREX). This system allows easy manipulation of distinct aspects of chromatin assembly such as post-translational histone modifications, the levels of histone chaperones and the concentration of distinct DNA binding factors. In total, 480 proteins have been quantified as chromatin enriched factors and their binding kinetics have been monitored in the time course of 15 min, 1 h and 4 h of chromatin assembly. The data accompanying the manuscript on this approach, Völker-Albert et al., 2016 "A quantitative proteomic analysis of in vitro assembled chromatin" [1], has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier submission number PRIDE: PXD002537 and PRIDE: PXD003445.

  6. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants.

    Science.gov (United States)

    Greenwood, Edward Jd; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick Jh; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-09-30

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.

  7. 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.

  8. Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

    Science.gov (United States)

    Patella, Francesca; Schug, Zachary T; Persi, Erez; Neilson, Lisa J; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-03-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

  9. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    Science.gov (United States)

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. 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.

  12. Proteomic Analysis Reveals That Metabolic Flows Affect the Susceptibility of Aeromonas hydrophila to Antibiotics

    Science.gov (United States)

    Yao, Zujie; Li, Wanxin; Lin, Yi; Wu, Qian; Yu, Feifei; Lin, Wenxiong; Lin, Xiangmin

    2016-01-01

    The overuse of antibiotics results in the development of antibiotic resistance and limits the useful life of these drugs in fighting bacteria, including Aeromonas hydrophila, a well-known opportunistic pathogen that causes serious infections in fish and other animals. In this study, we investigated the adaptive resistance mechanism in A. hydrophila by multiple proteomic methods. Dimethyl labeling and label-free methods were performed to compare the differential expression of proteins in response to various doses of oxytetracycline (OXY). The results point to the conclusions that, in response to OXY stress, translational processes increase the abundance of these proteins whereas largely central metabolic pathways decrease their abundance. To confirm our hypothesis, various exogenous metabolites were compounded with OXY, and the resulting survival capabilities were measured. Results show that 7 metabolites (malic acid, serine, methionine, etc.) significantly decreased the survival capabilities of A. hydrophila in the presence of OXY, whereas 4 metabolites (arginine, lysine, tyrosine, etc.) did the opposite. Further investigation suggests that a compound comprising exogenous metabolites in combination with various antibiotics could have a significant bactericidal effect and might come into widespread use, especially together with tetracycline antibiotics. These findings may provide new clues to the antimicrobial treatment of A. hydrophila infection. PMID:27991550

  13. Metabolic pathways of the wheat (Triticum aestivum endosperm amyloplast revealed by proteomics

    Directory of Open Access Journals (Sweden)

    Dupont Frances M

    2008-04-01

    Full Text Available Abstract Background By definition, amyloplasts are plastids specialized for starch production. However, a proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86 endosperm at 10 days after anthesis (DPA detected enzymes from many other metabolic and biosynthetic pathways. To better understand the role of amyloplasts in food production, the data from that study were evaluated in detail and an amyloplast metabolic map was outlined. Results Analysis of 288 proteins detected in an amyloplast preparation predicted that 178 were amyloplast proteins. Criteria included homology with known plastid proteins, prediction of a plastid transit peptide for the wheat gene product or a close homolog, known plastid location of the pathway, and predicted plastid location for other members of the same pathway. Of these, 135 enzymes were arranged into 18 pathways for carbohydrate, lipid, amino acid, nucleic acid and other biosynthetic processes that are critical for grain-fill. Functions of the other proteins are also discussed. Conclusion The pathways outlined in this paper suggest that amyloplasts play a central role in endosperm metabolism. The interacting effects of genetics and environment on starch and protein production may be mediated in part by regulatory mechanisms within this organelle.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation.

    Science.gov (United States)

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J; Zhang, Jianyi; Ge, Ying

    2015-08-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function.

  17. Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability*

    Science.gov (United States)

    Patella, Francesca; Schug, Zachary T.; Persi, Erez; Neilson, Lisa J.; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R.; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-01-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

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

    Science.gov (United States)

    Zhang, Qibin; Fillmore, Thomas L; Schepmoes, Athena A; Clauss, Therese R W; 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 that were significantly variant between those with type 1 diabetes (T1D) 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. 16 peptides were verified as having very good discriminating power, with areas under the receiver operating characteristic curve ≥ 0.8. Further validation with blinded serum samples from an independent cohort (10 healthy control and 10 type 1 diabetics) 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 sera from 50 age-matched type 2 diabetic individuals, and a subset of proteins, C1 inhibitor in particular, were exceptionally good discriminators between these two forms of diabetes. The panel of biomarkers distinguishing those with T1D from healthy controls and those with type 2 diabetes suggests that dysregulated innate immune responses may be associated with the development of this disorder.

  19. Proteomic analysis of saliva in HIV-positive heroin addicts reveals proteins correlated with cognition.

    Science.gov (United States)

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

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several 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+), 11 -negative (HIV-) heroin addicts. In addition, saliva samples were investigated from 11 HIV-, non-heroin addicted healthy controls. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV- heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. 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.

  20. Proteomic analysis of saliva in HIV-positive heroin addicts reveals proteins correlated with cognition.

    Directory of Open Access Journals (Sweden)

    Stephen S Dominy

    Full Text Available The prevalence of HIV-associated neurocognitive disorders (HAND remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several 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+, 11 -negative (HIV- heroin addicts. In addition, saliva samples were investigated from 11 HIV-, non-heroin addicted healthy controls. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV- heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. 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.

  1. Proteomics Analysis Reveals Post-Translational Mechanisms for Cold-Induced Metabolic Changes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Tian Li; Alma L.Burlingame; Zhi-Ping Deng; Zhi Yong Wang; Shou-Ling Xu; Juan A.Oses-Prieto; Sunita Putil; Peng Xu; Rui-Ju Wang; Kathy H.Li; David A.Malty; Liz-He An

    2011-01-01

    Cold-induced changes of gene expression and metabolism are critical for plants to survive freezing. Largely by changing gene expression, exposure to a period of non-freezing low temperatures increases plant tolerance to freezing-a phenomenon known as cold acclimation. Cold also induces rapid metabolic changes, which provide instant protection before temperature drops below freezing point. The molecular mechanisms for such rapid metabolic responses to cold remain largely unknown. Here, we use two-dimensional difference gel electrophoresis (2-D DIGE) analysis of sub-cellular fractions of Arabidopsis thaliana proteome coupled with spot identification by tandem mass spectrometry to identify early cold-responsive proteins in Arabidopsis. These proteins include four enzymes involved in starch degradation, three HSP100 proteins, several proteins in the tricarboxylic acid cycle, and sucrose metabolism. Upon cold treatment, the Disproportionating Enzyme 2 (DPE2), a cytosolic transglucosidase metabolizing maltose to glucose, increased rapidly in the centrifugation pellet fraction and decreased in the soluble fraction. Consistent with cold-induced inactivation of DPE2 enzymatic activity, the dpe2 mutant showed increased freezing tolerance without affecting the C-repeat binding transcription factor (CBF) transcriptional pathway. These results support a model that cold-induced inactivation of DPE2 leads to rapid accumulation of maltose, which is a cold-induced compatible solute that protects cells from freezing damage. This study provides evidence for a key role of rapid post-translational regulation of carbohydrate metabolic enzymes in plant protection against sudden temperature drop.

  2. Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence.

    Science.gov (United States)

    Kojima, Hisaya; Ogura, Yoshitoshi; Yamamoto, Nozomi; Togashi, Tomoaki; Mori, Hiroshi; Watanabe, Tomohiro; Nemoto, Fumiko; Kurokawa, Ken; Hayashi, Tetsuya; Fukui, Manabu

    2015-05-01

    Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8 Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of T. ingrica in lake sediment.

  3. Differential proteins of the optic ganglion in octopus vulgaris under methanol stress revealed using proteomics.

    Science.gov (United States)

    Huang, Lin; Huang, Qing-Yu; Chen, Hai-Bin; Huang, Fu-Sheng; Huang, He-Qing

    2011-10-01

    An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.

  4. Differential Proteomics of Urinary Exovesicles from Classical Galactosemic Patients Reveals Subclinical Kidney Insufficiency.

    Science.gov (United States)

    Staubach, Simon; Pekmez, Murat; Hanisch, Franz-Georg

    2016-06-03

    Classical galactosemia is caused by a nearly complete deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), resulting in a severely impaired galactose metabolism with galactose-1-phosphate and galactitol accumulation. Even on a galactose-restricted diet, patients develop serious long-term complications of the central nervous system and ovaries that may result from chronic cell-toxic effects exerted by endogenous galactose. To address the question of whether disease-associated cellular perturbations could affect the kidney function of the patients, we performed differential proteomics of detergent-resistant membranes from urinary exovesicles. Galactosemic samples (showing drastic shifts from high-mannose to complex-type N-glycosylation on exosomal N-glycoproteins) and healthy, sex-matched controls were analyzed in quadruplex iTRAQ experiments performed in biological and technical replicates. Particularly in the female patient group, the most striking finding was a drastic increase of abundant serum (glyco)proteins, like albumin, leucine-rich α-2-glycoprotein, fetuin, immunoglobulins, prostaglandin H2 d-isomerase, and α-1-microglobulin protein (AMBP), pointing to a subclinical failure of kidney filter function in galactosemic patients and resulting in a heavy overload of exosomal membranes with adsorbed serum (glyco)proteins. Several of these proteins are connected to TBMN and IgAN, proteinuria, and renal damage. The impairment of renal protein filtration was also indicated by increased protein contents derived from extracellular matrices and lysosomes.

  5. Proteomic analysis of pure human airway gland mucus reveals a large component of protective proteins.

    Directory of Open Access Journals (Sweden)

    Nam Soo Joo

    Full Text Available Airway submucosal glands contribute to innate immunity and protect the lungs by secreting mucus, which is required for mucociliary clearance and which also contains antimicrobial, anti-inflammatory, anti-proteolytic and anti-oxidant proteins. We stimulated glands in tracheal trimmings from three lung donors and collected droplets of uncontaminated mucus as they formed at the gland orifices under an oil layer. We analyzed the mucus using liquid chromatography-tandem mass spectrometry (LC-MS/MS. Analysis identified 5486 peptides and 441 proteins from across the 3 samples (269-319 proteins per subject. We focused on 269 proteins common to at least 2 0f 3 subjects, of which 102 (38% had protective or innate immunity functions. While many of these have long been known to play such roles, for many others their cellular protective functions have only recently been appreciated in addition to their well-studied biologic functions (e.g. annexins, apolipoproteins, gelsolin, hemoglobin, histones, keratins, and lumican. A minority of the identified proteins are known to be secreted via conventional exocytosis, suggesting that glandular secretion occurs via multiple mechanisms. Two of the observed protective proteins, major vault protein and prohibitin, have not been observed in fluid from human epithelial cultures or in fluid from nasal or bronchoalveolar lavage. Further proteomic analysis of pure gland mucus may help clarify how healthy airways maintain a sterile environment.

  6. Quantitative Proteomics Reveals That the Specific Methyltransferases Txr1p and Ezl2p Differentially Affect the Mono-, Di- and Trimethylation States of Histone H3 Lysine 27 (H3K27)*

    OpenAIRE

    Zhang, Chunchao; Molascon, Anthony J.; Gao, Shan; Liu, Yifan; Andrews, Philip C.

    2012-01-01

    Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using 15N metabolically labeled histones as the...

  7. Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit.

    Science.gov (United States)

    Kobori, Toshiro; Kodama, Mami; Hizume, Kohji; Yoshimura, Shige H; Ohtani, Toshio; Takeyasu, Kunio

    2006-01-01

    Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the 'on-substrate' lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, approximately 40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80-100 nm. Thus, these results suggest that the approximately 40 nm fiber would be a stable structural unit and fold the 80-100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.

  8. Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar

    Directory of Open Access Journals (Sweden)

    Lawrence Amanda M

    2010-11-01

    Full Text Available Abstract Background Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development. Results We report that poplar (Populus spp. has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast. Conclusion These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.

  9. Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics.

    Science.gov (United States)

    Nikolovski, Nino; Rubtsov, Denis; Segura, Marcelo P; Miles, Godfrey P; Stevens, Tim J; Dunkley, Tom P J; Munro, Sean; Lilley, Kathryn S; Dupree, Paul

    2012-10-01

    The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

  10. 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.

  11. Proteomic Analysis Reveals Proteins Involved in Seed Imbibition under Salt Stress in Rice.

    Science.gov (United States)

    Xu, Enshun; Chen, Mingming; He, Hui; Zhan, Chengfang; Cheng, Yanhao; Zhang, Hongsheng; Wang, Zhoufei

    2016-01-01

    Enhancement of salinity tolerance during seed germination is very important for direct seeding in rice. In this study, the salt-tolerant japonica landrace Jiucaiqing was used to determine the regulators that are involved in seed imbibition under salt stress. Briefly, the comparative proteomic analysis was conducted between dry (0 h) and imbibed (24 h) seeds with 150 mM NaCl. Under salt stress, the uptake of water increased rapidly before 24 h imbibition (Phase I), followed by a plateau of seed imbibition from 24 to 96 h imbibition (Phase II). We identified 14 proteins involved in seed imbibition, in which the majority of these proteins were involved in energy supply and storage protein. The early imbibition process was mediated by protein catabolism; the most of proteins were down-regulated after 24 h imbibition. Eleven genes in salt stress treated seeds were expressed early during the seed imbibition in comparison to control seeds. By comparison, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BPM), glutelin (GLU2.2 and GLU2.3), glucose-1-phosphate adenylyltransferase large subunit (GAS8), and cupin domain containing protein (CDP3.1 and CDP3.2) were near the regions of quantitative trait loci (QTLs) for seed dormancy, seed reserve utilization, and seed germination in Jiucaiqing. In particular, CDP3.1 was co-located in the region of qIR-3 for imbibition rate, and qGP-3 for germination percentage. The role of CDP3.1 was verified in enhancing seed germination under salt stress using T-DNA mutant. The identified proteins might be applicable for the improvement of seed germination under salt stress in rice.

  12. 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.

  13. Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I.

    Science.gov (United States)

    Guo, Tianyao; Duan, Zhigui; Chen, Jia; Xie, Chunliang; Wang, Ying; Chen, Ping; Wang, Xianchun

    2017-01-01

    Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.

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

    Science.gov (United States)

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

    2016-06-01

    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 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

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O.

    Science.gov (United States)

    Scott, Nichollas E; Marzook, N Bishara; Cain, Joel A; Solis, Nestor; Thaysen-Andersen, Morten; Djordjevic, Steven P; Packer, Nicolle H; Larsen, Martin R; Cordwell, Stuart J

    2014-11-07

    Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

  17. 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

  18. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    Science.gov (United States)

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  19. Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators.

    Directory of Open Access Journals (Sweden)

    So Yeon Kwon

    2016-04-01

    Full Text Available NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.

  20. Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation.

    Science.gov (United States)

    Zhang, Qiang; Cundiff, Judy K; Maria, Sarah D; McMahon, Robert J; Woo, Jessica G; Davidson, Barbara S; Morrow, Ardythe L

    2013-09-03

    In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study.

  1. Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation

    Directory of Open Access Journals (Sweden)

    Qiang Zhang

    2013-09-01

    Full Text Available In-depth understanding of the changing functions of human milk (HM proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study.

  2. Comparative proteomic analysis of a sea urchin (Heliocidaris erythrogramma) antibacterial response revealed the involvement of apextrin and calreticulin.

    Science.gov (United States)

    Dheilly, Nolwenn M; Haynes, Paul A; Bove, Ulysse; Nair, Sham V; Raftos, David A

    2011-02-01

    Echinoderms evolved early in the deuterostome lineage, and as such constitute model organisms for comparative physiology and immunology. The sea urchin genome sequence (Strongylocentrotus purpuratus) revealed a complex repertoire of genes with similarities to the immune response genes of other species. To complement these genomic data, we investigated the responses of sea urchins to the injection of bacteria using a comparative proteomics approach on a closely related species. In the sea urchin, Heliocidaris erythrogramma, the relative abundance of many proteins was altered in response to the injection of both bacteria and saline, suggesting their involvement in wounding responses, while others were differentially altered in response to bacteria only. The identities of 15 proteins that differed in relative abundance were determined by mass spectrometry. These proteins revealed a significant modification in energy metabolism in coelomocytes towards the consumption of glutamate and the production of NADPH after injection, as well as an increased concentration of cell signalling molecules, such as heterotrimeric guanine nucleotide-binding protein. The injection of bacteria specifically increased the abundance of apextrin and calreticulin, suggesting that these two proteins are involved in the sequestration or inactivation of bacteria.

  3. Motif decomposition of the phosphotyrosine proteome reveals a new N-terminal binding motif for SHIP2

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Hanke, S.; Hinsby, A. M.;

    2008-01-01

    Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data...

  4. Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and polycomb proteins in genome organization

    NARCIS (Netherlands)

    Denholtz, M.; Bonora, G.; Chronis, C.; Splinter, E.; de Laat, W.; Ernst, J.; Pellegrini, M.; Plath, K.

    2013-01-01

    The relationship between 3D organization of the genome and gene-regulatory networks is poorly understood. Here, we examined long-range chromatin interactions genome-wide in mouse embryonic stem cells (ESCs), iPSCs, and fibroblasts and uncovered a pluripotency-specific genome organization that is gra

  5. Characterization of genome-wide enhancer-promoter interactions reveals co-expression of interacting genes and modes of higher order chromatin organization

    Institute of Scientific and Technical Information of China (English)

    Iouri Chepelev; Gang Wei; Dara Wangsa; Qingsong Tang; Keji Zhao

    2012-01-01

    Recent epigenomic studies have predicted thousands of potential enhancers in the human genome.However,there has not been systematic characterization of target promoters for these potential enhancers.Using H3K4me2 as a mark for active enhancers,we identified genome-wide EP interactions in human CD4+ T cells.Among the 6 520 longdistance chromatin interactions,we identify 2 067 enhancers that interact with 1 619 promoters and enhance their expression.These enhancers exist in accessible chromatin regions and are associated with various histone modifications and polymerase Ⅱ binding.The promoters with interacting enhancers are expressed at higher levels than those without interacting enhancers,and their expression levels are positively correlated with the number of interacting enhancers.Interestingly,interacting promoters are co-expressed in a tissue-specific manner.We also find that chromosomes are organized into multiple levels of interacting domains.Our results define a global view of EP interactions and provide a data set to further understand mechanisms of enhancer targeting and long-range chromatin organization.The Gene Expression Omnibus accession number for the raw and analyzed chromatin interaction data is GSE32677.

  6. Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis

    DEFF Research Database (Denmark)

    Poulsen, Nina Aagaard; Andersen, Vibeke; Moller, Jens Christian;

    2012-01-01

    Background: Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa...... from patients with acute UC using mass spectrometry-based proteomic analysis. Methods: Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole...... annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon...

  7. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity.

    Science.gov (United States)

    Watson, Eleanor; Sherry, Aileen; Inglis, Neil F; Lainson, Alex; Jyothi, Dushyanth; Yaga, Raja; Manson, Erin; Imrie, Lisa; Everest, Paul; Smith, David G E

    2014-09-01

    Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  8. Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Hironori Waki

    2011-10-01

    Full Text Available Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type-specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq. FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA-mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our

  9. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity

    Directory of Open Access Journals (Sweden)

    Eleanor Watson

    2014-09-01

    Full Text Available Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC–ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith–Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  10. Quantitative shotgun proteomics reveals extensive changes to the proteome of the orbitofrontal cortex in rats that are hyperactive following withdrawal from a high sugar diet.

    Science.gov (United States)

    Franklin, Jane L; Mirzaei, Mehdi; Wearne, Travis A; Sauer, Melanie K; Homewood, Judi; Goodchild, Ann K; Haynes, Paul A; Cornish, Jennifer L

    2016-02-01

    In most Westernized societies, there has been an alarming increase in the consumption of sugar-sweetened drinks. For many adults these drinks represent a substantial proportion of their total daily caloric intake. Here we investigated whether extended exposure to sugar changes behavior and protein expression in the orbitofrontal cortex (OFC). Male adult Sprague-Dawley rats (n = 8 per group) were treated for 26 days with either water or a 10% sucrose solution. Locomotor behavior was measured on the first and last day of treatment, then 1 week after treatment. Following the 1-week period free from treatment, sucrose treated rats were significantly more active than the control. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free quantitative shotgun proteomic analyses of three rats from each group found 290 proteins were differentially expressed in the sucrose treated group when compared to the control group. Major changes in the proteome were seen in proteins related to energy metabolism, mitochondrial function and the cellular response to stress. This research does not seek to suggest that sugar will cause specific neurological disorders, however similar changes in proteins have been seen in neurological disorders such as Alzheimer's disease, Parkinson's disease and schizophrenia.

  11. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    Science.gov (United States)

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the “histone code.” We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to “canonical” chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living

  12. Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

    Directory of Open Access Journals (Sweden)

    Silvia Rodríguez-Lombardero

    2014-07-01

    Full Text Available Sky1 is the only member of the SR (Serine–Arginine protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

  13. Proteomics analysis of EV71-infected cells reveals the involvement of host protein NEDD4L in EV71 replication.

    Science.gov (United States)

    Kuo, Rei-Lin; Lin, Ya-Han; Wang, Robert Yung-Liang; Hsu, Chia-Wei; Chiu, Yi-Ting; Huang, Hsing-I; Kao, Li-Ting; Yu, Jau-Song; Shih, Shin-Ru; Wu, Chih-Ching

    2015-04-01

    Enterovirus 71 (EV71) is a human enterovirus that has seriously affected the Asia-Pacific area for the past two decades. EV71 infection can result in mild hand-foot-and-mouth disease and herpangina and may occasionally lead to severe neurological complications in children. However, the specific biological processes that become altered during EV71 infection remain unclear. To further explore host responses upon EV71 infection, we identified proteins differentially expressed in EV71-infected human glioblastoma SF268 cells using isobaric mass tag (iTRAQ) labeling coupled with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). Network analysis of proteins altered in cells infected with EV71 revealed that the changed biological processes are related to protein and ion transport, regulation of protein degradation, and homeostatic processes. We confirmed that the levels of NEDD4L and PSMF1 were increased and reduced, respectively, in EV71-infected cells compared to mock-infected control cells. To determine the physiological relevance of our findings, we investigated the consequences of EV71 infection in cells with NEDD4L or PSMF1 depletion. We found that the depletion of NEDD4L significantly reduced the replication of EV71, whereas PSMF1 knockdown enhanced EV71 replication. Collectively, our findings provide the first evidence of proteome-wide dysregulation by EV71 infection and suggest a novel role for the host protein NEDD4L in the replication of this virus.

  14. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress

    Science.gov (United States)

    Shi, Jianzhi; Chen, Yuting; Xu, Yan; Ji, Dehua; Chen, Changsheng; Xie, Chaotian

    2017-01-01

    Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress. PMID:28303955

  15. 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.

  16. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    Science.gov (United States)

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses.

  17. Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages.

    Directory of Open Access Journals (Sweden)

    Carmen Vogt

    Full Text Available Superparamagnetic iron oxide nanoparticles (SPIONs have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.

  18. Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages.

    Science.gov (United States)

    Vogt, Carmen; Pernemalm, Maria; Kohonen, Pekka; Laurent, Sophie; Hultenby, Kjell; Vahter, Marie; Lehtiö, Janne; Toprak, Muhammet S; Fadeel, Bengt

    2015-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated versus dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.

  19. Integrative proteome analysis of Brachypodium distachyon roots and leaves reveals a synergetic responsive network under H2O2 stress.

    Science.gov (United States)

    Bian, Yan-Wei; Lv, Dong-Wen; Cheng, Zhi-Wei; Gu, Ai-Qin; Cao, Hui; Yan, Yue-Ming

    2015-10-14

    The plant oxidative stress response is vital for defense against various abiotic and biotic stresses. In this study, ultrastructural changes and the proteomic response to H2O2 stress in roots and leaves of the model plant Brachypodium distachyon were studied. Transmission electron microscopy (TEM) showed that the ultrastructural damage in roots was more serious than in leaves. Particularly, the ultrastructures of organelles and the nucleus in root tip cells were damaged, leading to the inhibition of normal biological activities of roots, which then spread throughout the plant. Based on two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS, 84 and 53 differentially accumulated protein (DAP) spots representing 75 and 45 unique proteins responsive to H2O2 stress in roots and leaves, respectively, were identified. These protein species were mainly involved in signal transduction, energy metabolism, redox homeostasis/stress defense, protein folding/degradation, and cell wall/cell structure. Interestingly, two 14-3-3 proteins (GF14-B and GF14-D) were identified as DAPs in both roots and leaves. Protein-protein interaction (PPI) analysis revealed a synergetic H2O2-responsive network.

  20. 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...... 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...

  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.

    2017-01-01

    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

  2. Stimulatory effect of Echinacea purpurea extract on the trafficking activity of mouse dendritic cells: revealed by genomic and proteomic analyses

    Directory of Open Access Journals (Sweden)

    Wang Bi-Xue

    2010-11-01

    Full Text Available Abstract Background Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF of a stem and leaf (S+L extract of E. purpurea ([BF/S+L/Ep] containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs were analyzed using primary cultures. Results Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs. Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8, or were chemokines (Cxcl2, Cxcl7 or signaling molecules (Nrxn1, Pkce and Acss1. TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups. Conclusion Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical

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

    Directory of Open Access Journals (Sweden)

    Linghua Chen

    Full Text Available 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

  4. 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.

  5. 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...

  6. 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...

  7. Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping.

    Science.gov (United States)

    Kernohan, Kristin D; Vernimmen, Douglas; Gloor, Gregory B; Bérubé, Nathalie G

    2014-07-01

    ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure.

  8. Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways.

    Science.gov (United States)

    Müller, Sebastian; Balaz, Miroslav; Stefanicka, Patrik; Varga, Lukas; Amri, Ez-Zoubir; Ukropec, Jozef; Wollscheid, Bernd; Wolfrum, Christian

    2016-07-15

    Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype.

    Science.gov (United States)

    Klemmer, Patricia; Meredith, Rhiannon M; Holmgren, Carl D; Klychnikov, Oleg I; Stahl-Zeng, Jianru; Loos, Maarten; van der Schors, Roel C; Wortel, Joke; de Wit, Heidi; Spijker, Sabine; Rotaru, Diana C; Mansvelder, Huibert D; Smit, August B; Li, Ka Wan

    2011-07-22

    Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

  11. Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy.

    Science.gov (United States)

    Cesnik, Anthony J; Shortreed, Michael R; Sheynkman, Gloria M; Frey, Brian L; Smith, Lloyd M

    2016-03-04

    Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Regulation of BZR1 in fruit ripening revealed by iTRAQ proteomics analysis

    Science.gov (United States)

    Liu, Lihong; Liu, Haoran; Li, Shuo; Zhang, Xin; Zhang, Min; Zhu, Ning; Dufresne, Craig P.; Chen, Sixue; Wang, Qiaomei

    2016-01-01

    Fruit ripening is a complex and genetically programmed process. Brassinosteroids (BRs) play an essential role in plant growth and development, including fruit ripening. As a central component of BR signaling, the transcription factor BZR1 is involved in fruit development in tomato. However, the transcriptional network through which BZR1 regulates fruit ripening is mostly unknown. In this study, we use isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology to explore important proteins regulated by BZR1 in two independent tomato transgenic lines over-expressing BZR1-1D at four ripening stages, identifying 411 differentially expressed proteins. These proteins were implicated in light reaction, plant hormone pathways and cell-wall-related metabolism, etc. The ‘light reaction’ metabolic pathway was identified as a markedly enhanced pathway by BZR1 during tomato fruit ripening. The protein level of a probable 2-oxoglutarate-dependent dioxygenase 2-ODD2, involved in gibberellin biosynthesis was significantly increased at all four developmental and ripening stages. The results reveal molecular links between BR signaling pathway and downstream components involved in multiple ripening-associated events during tomato fruit ripening, which will provide new insights into the molecular mechanisms underlying tomato ripening regulatory networks, and be potential in understanding BR-regulated fruit ripening. PMID:27680870

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

    Directory of Open Access Journals (Sweden)

    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

  17. Single Molecule Studies of Chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Jeans, C; Thelen, M P; Noy, A

    2006-02-06

    In eukaryotic cells, DNA is packaged as chromatin, a highly ordered structure formed through the wrapping of the DNA around histone proteins, and further packed through interactions with a number of other proteins. In order for processes such as DNA replication, DNA repair, and transcription to occur, the structure of chromatin must be remodeled such that the necessary enzymes can access the DNA. A number of remodeling enzymes have been described, but our understanding of the remodeling process is hindered by a lack of knowledge of the fine structure of chromatin, and how this structure is modulated in the living cell. We have carried out single molecule experiments using atomic force microscopy (AFM) to study the packaging arrangements in chromatin from a variety of cell types. Comparison of the structures observed reveals differences which can be explained in terms of the cell type and its transcriptional activity. During the course of this project, sample preparation and AFM techniques were developed and optimized. Several opportunities for follow-up work are outlined which could provide further insight into the dynamic structural rearrangements of chromatin.

  18. Chromatin is wonderful stuff.

    NARCIS (Netherlands)

    R. van Driel

    2007-01-01

    Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all contro

  19. 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.

  20. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

    Science.gov (United States)

    Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl

    2015-06-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

  1. Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins.

    Science.gov (United States)

    Mesner, Larry D; Valsakumar, Veena; Cieslik, Marcin; Pickin, Rebecca; Hamlin, Joyce L; Bekiranov, Stefan

    2013-11-01

    We have devised a method for isolating virtually pure and comprehensive libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo. We have now sequenced and mapped the bubble-containing fragments from GM06990, a near-normal EBV-transformed lymphoblastoid cell line, and have compared origin distributions with a comprehensive replication timing study recently published for this cell line. We find that early-firing origins, which represent ∼32% of all origins, overwhelmingly represent zones, associate only marginally with active transcription units, are localized within large domains of open chromatin, and are significantly associated with DNase I hypersensitivity. Origin "density" falls from early- to mid-S-phase, but rises again in late S-phase to levels only 17% lower than in early S-phase. Unexpectedly, late origin density calculated on the 1-Mb scale increases as a function of increasing chromatin compaction. Furthermore, the median efficiency of origins in late-replicating, heterochromatic domains is only 25% lower than in early-replicating euchromatic loci. Thus, the activation of early- and late-firing origins must be regulated by quintessentially different mechanisms. The aggregate data can be unified into a model in which initiation site selection is driven almost entirely by epigenetic factors that fashion both the long-range and local chromatin environments, with underlying DNA sequence and local transcriptional activity playing only minor roles. Importantly, the comprehensive origin map we have prepared for GM06990 overlaps moderately well with origin maps recently reported for the genomes of four different human cell lines based on the distributions of small nascent strands.

  2. Integrated Proteomics and Genomics Analysis Reveals a Novel Mesenchymal to Epithelial Reverting Transition in Leiomyosarcoma through Regulation of Slug*

    Science.gov (United States)

    Yang, Jilong; Eddy, James A.; Pan, Yuan; Hategan, Andrea; Tabus, Ioan; Wang, Yingmei; Cogdell, David; Price, Nathan D.; Pollock, Raphael E.; Lazar, Alexander J. F.; Hunt, Kelly K.; Trent, Jonathan C.; Zhang, Wei

    2010-01-01

    Leiomyosarcoma is one of the most common mesenchymal tumors. Proteomics profiling analysis by reverse-phase protein lysate array surprisingly revealed that expression of the epithelial marker E-cadherin (encoded by CDH1) was significantly elevated in a subset of leiomyosarcomas. In contrast, E-cadherin was rarely expressed in the gastrointestinal stromal tumors, another major mesenchymal tumor type. We further sought to 1) validate this finding, 2) determine whether there is a mesenchymal to epithelial reverting transition (MErT) in leiomyosarcoma, and if so 3) elucidate the regulatory mechanism responsible for this MErT. Our data showed that the epithelial cell markers E-cadherin, epithelial membrane antigen, cytokeratin AE1/AE3, and pan-cytokeratin were often detected immunohistochemically in leiomyosarcoma tumor cells on tissue microarray. Interestingly, the E-cadherin protein expression was correlated with better survival in leiomyosarcoma patients. Whole genome microarray was used for transcriptomics analysis, and the epithelial gene expression signature was also associated with better survival. Bioinformatics analysis of transcriptome data showed an inverse correlation between E-cadherin and E-cadherin repressor Slug (SNAI2) expression in leiomyosarcoma, and this inverse correlation was validated on tissue microarray by immunohistochemical staining of E-cadherin and Slug. Knockdown of Slug expression in SK-LMS-1 leiomyosarcoma cells by siRNA significantly increased E-cadherin; decreased the mesenchymal markers vimentin and N-cadherin (encoded by CDH2); and significantly decreased cell proliferation, invasion, and migration. An increase in Slug expression by pCMV6-XL5-Slug transfection decreased E-cadherin and increased vimentin and N-cadherin. Thus, MErT, which is mediated through regulation of Slug, is a clinically significant phenotype in leiomyosarcoma. PMID:20651304

  3. Proteomic analysis of the excretory-secretory products from larval stages of Ascaris suum reveals high abundance of glycosyl hydrolases.

    Directory of Open Access Journals (Sweden)

    Tao Wang

    Full Text Available BACKGROUND: Ascaris lumbricoides and Ascaris suum are socioeconomically important and widespread parasites of humans and pigs, respectively. The excretory-secretory (ES molecules produced and presented at the parasite-host interface during the different phases of tissue invasion and migration are likely to play critical roles in the induction and development of protective immune and other host responses. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify the ES proteins of the different larval stages (L3-egg, L3-lung and L4 by LC-MS/MS. In total, 106 different proteins were identified, 20 in L3-egg, 45 in L3-lung stage and 58 in L4. Although most of the proteins identified were stage-specific, 15 were identified in the ES products of at least two stages. Two proteins, i.e. a 14-3-3-like protein and a serpin-like protein, were present in the ES products from the three different larval stages investigated. Interestingly, a comparison of ES products from L4 with those of L3-egg and L3-lung showed an abundance of metabolic enzymes, particularly glycosyl hydrolases. Further study indicated that most of these glycolytic enzymes were transcriptionally upregulated from L4 onwards, with a peak in the adult stage, particularly in intestinal tissue. This was also confirmed by enzymatic assays, showing the highest glycosidase activity in protein extracts from adult worms gut. CONCLUSIONS/SIGNIFICANCE: The present proteomic analysis provides important information on the host-parasite interaction and the biology of the migratory stages of A. suum. In particular, the high transcriptional upregulation of glycosyl hydrolases from the L4 stage onwards reveals that the degradation of complex carbohydrates forms an essential part of the energy metabolism of this parasite once it establishes in the small intestine.

  4. Chromatin Structure and Function

    CERN Document Server

    Wolffe, Alan P

    1999-01-01

    The Third Edition of Chromatin: Structure and Function brings the reader up-to-date with the remarkable progress in chromatin research over the past three years. It has been extensively rewritten to cover new material on chromatin remodeling, histone modification, nuclear compartmentalization, DNA methylation, and transcriptional co-activators and co-repressors. The book is written in a clear and concise fashion, with 60 new illustrations. Chromatin: Structure and Function provides the reader with a concise and coherent account of the nature, structure, and assembly of chromatin and its active

  5. Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

    Science.gov (United States)

    Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

    2015-07-01

    Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

  6. Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria.

    Science.gov (United States)

    Gomes, Douglas Fabiano; Batista, Jesiane Stefania da Silva; Torres, Adalgisa Ribeiro; de Souza Andrade, Diva; Galli-Terasawa, Lygia Vitoria; Hungria, Mariangela

    2012-03-01

    Rhizobium tropici strain PRF 81 is used in commercial inoculants for common-bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two-dimensional gel electrophoresis and MALDI-TOF/TOF-TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia-legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.

  7. Chromatin Remodelers: From Function to Dysfunction

    Directory of Open Access Journals (Sweden)

    Gernot Längst

    2015-06-01

    Full Text Available Chromatin remodelers are key players in the regulation of chromatin accessibility and nucleosome positioning on the eukaryotic DNA, thereby essential for all DNA dependent biological processes. Thus, it is not surprising that upon of deregulation of those molecular machines healthy cells can turn into cancerous cells. Even though the remodeling enzymes are very abundant and a multitude of different enzymes and chromatin remodeling complexes exist in the cell, the particular remodeling complex with its specific nucleosome positioning features must be at the right place at the right time in order to ensure the proper regulation of the DNA dependent processes. To achieve this, chromatin remodeling complexes harbor protein domains that specifically read chromatin targeting signals, such as histone modifications, DNA sequence/structure, non-coding RNAs, histone variants or DNA bound interacting proteins. Recent studies reveal the interaction between non-coding RNAs and chromatin remodeling complexes showing importance of RNA in remodeling enzyme targeting, scaffolding and regulation. In this review, we summarize current understanding of chromatin remodeling enzyme targeting to chromatin and their role in cancer development.

  8. The chemosensory appendage proteome of Amblyomma americanum (Acari: Ixodidae) reveals putative odorant-binding and other chemoreception-related proteins

    Science.gov (United States)

    Proteomic analyses were done on 2 chemosensory appendages of the lone star tick, Amblyomma americanum. Proteins in the fore tarsi, which contain the olfactory Haller's organ, and in the palps, that include gustatory sensilla, were compared with proteins in the third tarsi. Also, male and female tick...

  9. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2

    NARCIS (Netherlands)

    Stierum, R.; Gaspari, M.; Dommels, Y.; Ouatas, T.; Pluk, H.; Jespersen, S.; Vogels, J.; Verhoeckx, K.; Groten, J.; Ommen, B. van

    2003-01-01

    Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvi

  10. Plasma metabolomics and proteomics profiling after a postprandial challenge reveal subtle diet effects on human metabolic status

    NARCIS (Netherlands)

    Pellis, L.; Erk, M.J. van; Ommen, B. van; Bakker, G.C.M.; Hendriks, H.F.J.; Cnubben, N.H.P.; Kleemann, R.; Someren, E.P. van; Bobeldijk, I.; Rubingh, C.M.; Wopereis, S.

    2012-01-01

    We introduce the metabolomics and proteomics based Postprandial Challenge Test (PCT) to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The PCT comprised consumption of a standardized 500 ml dairy shake containing respectively 59, 30 and 12 ener

  11. Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

    Science.gov (United States)

    Andrade-Silva, Débora; Zelanis, André; Kitano, Eduardo S; Junqueira-de-Azevedo, Inácio L M; Reis, Marcelo S; Lopes, Aline S; Serrano, Solange M T

    2016-08-05

    Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

  12. 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.

  13. Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling.

    Science.gov (United States)

    Anderson, Johnathon D; Johansson, Henrik J; Graham, Calvin S; Vesterlund, Mattias; Pham, Missy T; Bramlett, Charles S; Montgomery, Elizabeth N; Mellema, Matt S; Bardini, Renee L; Contreras, Zelenia; Hoon, Madeline; Bauer, Gerhard; Fink, Kyle D; Fury, Brian; Hendrix, Kyle J; Chedin, Frederic; El-Andaloussi, Samir; Hwang, Billie; Mulligan, Michael S; Lehtiö, Janne; Nolta, Jan A

    2016-03-01

    Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.

  14. 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.

  15. Comparative Analysis of Genomics and Proteomics in the New Isolated Bacillus thuringiensis X022 Revealed the Metabolic Regulation Mechanism of Carbon Flux Following Cu(2+) Treatment.

    Science.gov (United States)

    Quan, Meifang; Xie, Junyan; Liu, Xuemei; Li, Yang; Rang, Jie; Zhang, Tong; Zhou, Fengjuan; Xia, Liqiu; Hu, Shengbiao; Sun, Yunjun; Ding, Xuezhi

    2016-01-01

    Bacillus thuringiensis (Bt) X022 is a novel strain isolated from soil in China, and showed strong insecticidal activity against several Lepidopteran pests. In this work, we performed whole genome sequencing of this Bt strain using the next-generation sequencing technology, and further conducted a comparative analysis with the proteomics data of the specific spore-release period based on LC-MS/MS approach. The Bt X022 genome consisted of one circular chromosomal DNA and seven plasmids, which were further functionally annotated using the RAST server. Comparative analysis of insecticidal substances showed that X022 contained genes coding for three Cry proteins (Cry1Ac, Cry1Ia and Cry2Ab) and a vegetative insecticidal protein (Vip3A). However, three insecticidal crystal proteins (ICPs) (Cry1Ca, Cry1Ac and Cry1Da) were detected by proteomics in the spore-release period. Moreover, a putative biosynthetic gene cluster and the metabolic pathway for poly-β-hydroxybutyrate in Bt X022 were deduced based on the comparative analysis of genomic and proteomic data, which revealed the metabolic regulation mechanism of carbon flux correlated with increased production of ICPs caused by Cu(2+.) Hence, these results provided a deeper understanding of the genetic background and protein expression profile of Bt X022. This study established a foundation for directed genetic modification and further application of this new isolated Bt strain.

  16. Comparative analysis of genomics and proteomics in the new isolated Bacillus thuringiensis X022 revealed the metabolic regulation mechanism of carbon flux following Cu2+ treatment

    Directory of Open Access Journals (Sweden)

    Meifang eQuan

    2016-05-01

    Full Text Available Bacillus thuringiensis (Bt X022, which showed strong insecticidal activity against several Lepidopteran pests, is a novel strain isolated from soil in China. In this work, we performed whole genome sequencing of this Bt strain using the next-generation sequencing technology, and further conducted a comparative analysis with the proteomics data of the specific spore-release period based on LC-MS/MS approach. The Bt X022 genome consisted of one circular chromosomal DNA and seven plasmids, which were further functionally annotated using the RAST server. Comparative analysis of insecticidal substances showed that X022 contained genes coding for three Cry proteins (Cry1Ac, Cry1Ia and Cry2Ab and a vegetative insecticidal protein (Vip3A. However, three insecticidal crystal proteins (ICPs (Cry1Ca, Cry1Ac and Cry1Da were detected by proteomics in the spore-release period. Moreover, a putative biosynthetic gene cluster and the metabolic pathway for poly-β-hydroxybutyrate in Bt X022 were deduced based on the comparative analysis of genomic and proteomic data, which revealed the metabolic regulation mechanism of carbon flux correlated with increased production of ICPs caused by Cu2+ . Hence, these results provided a deeper understanding of the genetic background and protein expression profile of Bt X022. This study established a foundation for directed genetic modification and further application of this new isolated Bt strain.

  17. Comparative Analysis of Genomics and Proteomics in the New Isolated Bacillus thuringiensis X022 Revealed the Metabolic Regulation Mechanism of Carbon Flux Following Cu2+ Treatment

    Science.gov (United States)

    Quan, Meifang; Xie, Junyan; Liu, Xuemei; Li, Yang; Rang, Jie; Zhang, Tong; Zhou, Fengjuan; Xia, Liqiu; Hu, Shengbiao; Sun, Yunjun; Ding, Xuezhi

    2016-01-01

    Bacillus thuringiensis (Bt) X022 is a novel strain isolated from soil in China, and showed strong insecticidal activity against several Lepidopteran pests. In this work, we performed whole genome sequencing of this Bt strain using the next-generation sequencing technology, and further conducted a comparative analysis with the proteomics data of the specific spore-release period based on LC-MS/MS approach. The Bt X022 genome consisted of one circular chromosomal DNA and seven plasmids, which were further functionally annotated using the RAST server. Comparative analysis of insecticidal substances showed that X022 contained genes coding for three Cry proteins (Cry1Ac, Cry1Ia and Cry2Ab) and a vegetative insecticidal protein (Vip3A). However, three insecticidal crystal proteins (ICPs) (Cry1Ca, Cry1Ac and Cry1Da) were detected by proteomics in the spore-release period. Moreover, a putative biosynthetic gene cluster and the metabolic pathway for poly-β-hydroxybutyrate in Bt X022 were deduced based on the comparative analysis of genomic and proteomic data, which revealed the metabolic regulation mechanism of carbon flux correlated with increased production of ICPs caused by Cu2+. Hence, these results provided a deeper understanding of the genetic background and protein expression profile of Bt X022. This study established a foundation for directed genetic modification and further application of this new isolated Bt strain. PMID:27303381

  18. 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.

  19. A high-resolution tissue-specific proteome and phosphoproteome atlas of maize primary roots reveals functional gradients along the root axes.

    Science.gov (United States)

    Marcon, Caroline; Malik, Waqas Ahmed; Walley, Justin W; Shen, Zhouxin; Paschold, Anja; Smith, Laurie G; Piepho, Hans-Peter; Briggs, Steven P; Hochholdinger, Frank

    2015-05-01

    A high-resolution proteome and phosphoproteome atlas of four maize (Zea mays) primary root tissues, the cortex, stele, meristematic zone, and elongation zone, was generated. High-performance liquid chromatography coupled with tandem mass spectrometry identified 11,552 distinct nonmodified and 2,852 phosphorylated proteins across the four root tissues. Two gradients reflecting the abundance of functional protein classes along the longitudinal root axis were observed. While the classes RNA, DNA, and protein peaked in the meristematic zone, cell wall, lipid metabolism, stress, transport, and secondary metabolism culminated in the differentiation zone. Functional specialization of tissues is underscored by six of 10 cortex-specific proteins involved in flavonoid biosynthesis. Comparison of this data set with high-resolution seed and leaf proteome studies revealed 13% (1,504/11,552) root-specific proteins. While only 23% of the 1,504 root-specific proteins accumulated in all four root tissues, 61% of all 11,552 identified proteins accumulated in all four root tissues. This suggests a much higher degree of tissue-specific functionalization of root-specific proteins. In summary, these data illustrate the remarkable plasticity of the proteomic landscape of maize primary roots and thus provide a starting point for gaining a better understanding of their tissue-specific functions.

  20. Characterizing the nuclear proteome of Paracoccidioides spp.

    Science.gov (United States)

    Oliveira, Lucas Nojosa; Casaletti, Luciana; Báo, Sônia Nair; Borges, Clayton Luiz; de Sousa Lima, Patrícia; de Almeida Soares, Célia Maria

    2016-10-01

    Paracoccidioidomycosis is an endemic disease in Latin America, caused by thermo dimorphic fungi of the genus Paracoccidioides. Although previous proteome analyses of Paracoccidioides spp. have been carried out, the nuclear subproteome of this pathogen has not been described. In this way, we aimed to characterize the nuclear proteome of Paracoccidioides species, in the yeast form. For that, yeast cells were disrupted and submitted to cell fractionation. The purity of the nuclear fraction was confirmed by fluorescence and electron microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 867 proteins. In order to support our enrichment method for nuclear proteins, bioinformatics analysis were applied that allowed the identification of 281 proteins with nuclear localization. The analysis revealed proteins related to DNA maintenance, gene expression, synthesis and processing of messenger and ribosomal RNAs, likewise proteins of nuclear-cytoplasmic traffic. It was also possible to detect some proteins that are poorly expressed, like transcription factors involved in important roles such as resistance to abiotic stress, sporulation, cellular growth and DNA and chromatin maintenance. This is the first descriptive nuclear proteome of Paracoccidioides spp. that can be useful as an important platform base for fungi-specific nuclear processes.

  1. 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...... to H2O2 were typically related to carbohydrate and energy metabolism, cysteine biosynthesis, and stress. In particular, increased cysteine synthase activity may accumulate a cysteine pool relevant for protein stability, enzyme catalysis and the disulfide-reducing pathway. The stress response further...... by refolding. The proteome analysis provides novel insight into resistance mechanisms in lactic acid bacteria against reactive oxygen species and constitutes a valuable starting point for improving industrial processes, food design or strain engineering preserving microorganism viability....

  2. 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.

  3. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    Science.gov (United States)

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

  4. 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...

  5. Complementary transcriptomic and proteomic analyses of a chlorophyll-deficient tea plant cultivar reveal multiple metabolic pathway changes.

    Science.gov (United States)

    Wang, Lu; Cao, Hongli; Chen, Changsong; Yue, Chuan; Hao, Xinyuan; Yang, Yajun; Wang, Xinchao

    2016-01-01

    To uncover the mechanisms that underlie the chlorina phenotype of the tea plant, this study employs morphological, biochemical, transcriptomic, and iTRAQ-based proteomic analyses to compare the green tea cultivar LJ43 and the yellow-leaf tea cultivar ZH1. ZH1 exhibited the chlorina phenotype, with significantly decreased chlorophyll content and abnormal chloroplast development compared with LJ43. ZH1 also displayed higher theanine and free amino acid content and lower carotenoid and catechin content. Microarray and iTRAQ analyses indicated that the differentially expressed genes and proteins could be mapped to the following pathways: 'phenylpropanoid biosynthesis,' 'glutathione metabolism,' 'phenylalanine metabolism,' 'photosynthesis,' and 'flavonoid biosynthesis.' Altered gene and protein levels in these pathways may account for the increased amino acid content and reduced chlorophyll and flavonoid content of ZH1. Altogether, this study combines transcriptomic and proteomic approaches to better understand the mechanisms responsible for the chlorina phenotype.

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

    Institute of Scientific and Technical Information of China (English)

    Qiaosong Yang; Junhua Wu; Chunyu Li; Yuerong Wei; Ou Sheng; Chunhua Hu; Ruibin Kuang

    2012-01-01

    Banana and its close relative,plantain are globally important crops and there is of considerable interest in optimizing their cultivation.Plantain has superior cold tolerance compared to 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℃ and subsequently allowed to recover for 24 h at 28℃.A total of 3,477 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,while 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 7 differentially expressed,cold-response candidate plantain proteins in order to validate the proteomics data.Similar analyses of the 7 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 higher 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 proteomic analysis.

  7. Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics

    OpenAIRE

    Diamond, Deborah L.; Syder, Andrew J; Jacobs, Jon M.; Sorensen, Christina M.; Kathie-Anne Walters; Sean C Proll; Jason E. McDermott; Gritsenko, Marina A.; Qibin Zhang; Rui Zhao; Metz, Thomas O.; David G Camp; Waters, Katrina M.; Smith, Richard D.; Rice, Charles M.

    2010-01-01

    Author Summary As parasites, viruses rely on the cells they infect to provide the energy and building blocks required for their survival and propagation. However, relatively little is known about the extent to which viruses modulate host cell metabolism and the consequences of these disruptions. Here we integrate proteomic and lipidomic profiling with computational modeling approaches to probe the impact of HCV infection on the global metabolism of cultured hepatoma cells, and to understand t...

  8. 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

  9. Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures.

    Science.gov (United States)

    Johnston, Harvey E; Carter, Matthew J; Cox, Kerry L; Dunscombe, Melanie; Manousopoulou, Antigoni; Townsend, Paul A; Garbis, Spiros D; Cragg, Mark S

    2017-03-01

    Approximately 800,000 leukemia and lymphoma cases are diagnosed worldwide each year. Burkitt's lymphoma (BL) and chronic lymphocytic leukemia (CLL) are examples of contrasting B-cell cancers; BL is a highly aggressive lymphoid tumor, frequently affecting children, whereas CLL typically presents as an indolent, slow-progressing leukemia affecting the elderly. The B-cell-specific overexpression of the myc and TCL1 oncogenes in mice induce spontaneous malignancies modeling BL and CLL, respectively. Quantitative mass spectrometry proteomics and isobaric labeling were employed to examine the biology underpinning contrasting Eμ-myc and Eμ-TCL1 B-cell tumors. Additionally, the plasma proteome was evaluated using subproteome enrichment to interrogate biomarker emergence and the systemic effects of tumor burden. Over 10,000 proteins were identified (qIL5) receptor. IL5 treatment promoted Eμ-TCL1 tumor proliferation, suggesting an amplification of IL5-induced AKT signaling by TCL1. Tumor plasma contained a substantial tumor lysis signature, most prominent in Eμ-myc plasma, whereas Eμ-TCL1 plasma contained signatures of immune-response, inflammation and microenvironment interactions, with putative biomarkers in early-stage cancer. These findings provide a detailed characterization of contrasting B-cell tumor models, identifying common and specific tumor mechanisms. Integrated plasma proteomics allowed the dissection of a systemic response and a tumor lysis signature present in early- and late-stage cancers, respectively. Overall, this study suggests common B-cell cancer signatures exist and illustrates the potential of the further evaluation of B-cell cancer subtypes by integrative proteomics.

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

    Science.gov (United States)

    Chaubey, Pururawa Mayank; Hofstetter, Lia; Roschitzki, Bernd; Stieger, Bruno

    2016-01-01

    Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

  13. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

    Science.gov (United States)

    Procaccini, Claudio; Carbone, Fortunata; Di Silvestre, Dario; Brambilla, Francesca; De Rosa, Veronica; Galgani, Mario; Faicchia, Deriggio; Marone, Gianni; Tramontano, Donatella; Corona, Marco; Alviggi, Carlo; Porcellini, Antonio; La Cava, Antonio; Mauri, Pierluigi; Matarese, Giuseppe

    2016-01-01

    Summary Human CD4+CD25hiFoxp3+CD127− Treg and CD4+CD25−Foxp3− Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells. PMID:26885861

  14. 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.

  15. 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.

  16. Proteomic analyses of human cytomegalovirus strain AD169 derivatives reveal highly conserved patterns of viral and cellular proteins in infected fibroblasts.

    Science.gov (United States)

    Reyda, Sabine; Büscher, Nicole; Tenzer, Stefan; Plachter, Bodo

    2014-01-07

    Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.

  17. Phage-Induced Expression of CRISPR-Associated Proteins is Revealed by Shotgun Proteomics in Streptococcus thermophilus

    Energy Technology Data Exchange (ETDEWEB)

    Young, Jacque C [ORNL; Dill, Brian [ORNL; Pan, Chongle [ORNL; Hettich, Robert {Bob} L [ORNL; Banfield, Jillian F. [University of California, Berkeley; Shah, Manesh B [ORNL; Fremaux, Christophe [Danisco France SAS; Horvath, Philippe [Danisco France SAS; Barrangou, Rodolphe [Danisco USA; Verberkmoes, Nathan C [ORNL

    2012-01-01

    The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response: infection of S. thermophilus DGCC7710 with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectroscopy. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance during peak infection, including the Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Proteomic Analysis of the Rat Canalicular Membrane Reveals Expression of a Complex System of P4-ATPases in Liver.

    Directory of Open Access Journals (Sweden)

    Pururawa Mayank Chaubey

    Full Text Available Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.

  1. Functional proteomic analysis revealed ground-base ion radiations cannot reflect biological effects of space radiations of rice

    Science.gov (United States)

    Wang, Wei; Sun, Yeqing; Zhao, Qian; Han, Lu

    2016-07-01

    Highly ionizing radiation (HZE) in space is considered as main factor causing biological effects. Radiobiological studies during space flights are unrepeatable due to the variable space radiation environment, ground-base ion radiations are usually performed to simulate of the space biological effect. Spaceflights present a low-dose rate (0.1˜~0.3mGy/day) radiation environment inside aerocrafts while ground-base ion radiations present a much higher dose rate (100˜~500mGy/min). Whether ground-base ion radiation can reflect effects of space radiation is worth of evaluation. In this research, we compared the functional proteomic profiles of rice plants between on-ground simulated HZE particle radiation and spaceflight treatments. Three independent ground-base seed ionizing radiation experiments with different cumulative doses (dose range: 2˜~20000mGy) and different liner energy transfer (LET) values (13.3˜~500keV/μμm) and two independent seed spaceflight experiments onboard Chinese 20th satellite and SZ-6 spacecraft were carried out. Alterations in the proteome were analyzed by two-dimensional difference gel electrophoresis (2-D DIGE) with MALDI-TOF/TOF mass spectrometry identifications. 45 and 59 proteins showed significant (pmetabolic process, protein folding and phosphorylation. The results implied that ground-base radiations cannot truly reflect effects of spaceflight radiations, ground-base radiation was a kind of indirect effect to rice causing oxidation and metabolism stresses, but space radiation was a kind of direct effect leading to macromolecule (DNA and protein) damage and signal pathway disorders. This functional proteomic analysis work might provide a new evaluation method for further on-ground simulated HZE radiation experiments.

  2. Proteomics analysis of human obesity reveals the epigenetic factor HDAC4 as a potential target for obesity.

    Directory of Open Access Journals (Sweden)

    Mohamed Abu-Farha

    Full Text Available Sedentary lifestyle and excessive energy intake are prominent contributors to obesity; a major risk factors for the development of insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidating the molecular mechanisms underlying these chronic conditions is of relevant importance as it might lead to the identification of novel anti-obesity targets. The purpose of the current study is to investigate differentially expressed proteins between lean and obese subjects through a shot-gun quantitative proteomics approach using peripheral blood mononuclear cells (PBMCs extracts as well as potential modulation of those proteins by physical exercise. Using this approach, a total of 47 proteins showed at least 1.5 fold change between lean and obese subjects. In obese, the proteomic profiling before and after 3 months of physical exercise showed differential expression of 38 proteins. Thrombospondin 1 (TSP1 was among the proteins that were upregulated in obese subjects and then decreased by physical exercise. Conversely, the histone deacetylase 4 (HDAC4 was downregulated in obese subjects and then induced by physical exercise. The proteomic data was further validated by qRT-PCR, Western blot and immunohistochemistry in both PBMCs and adipose tissue. We also showed that HDAC4 levels correlated positively with maximum oxygen consumption (VO2 Max but negatively with body mass index, percent body fat, and the inflammatory chemokine RANTES. In functional assays, our data indicated that ectopic expression of HDAC4 significantly impaired TNF-α-dependent activation of NF-κB, establishing thus a link between HDAC4 and regulation of the immune system. Together, the expression pattern of HDAC4 in obese subjects before and after physical exercise, its correlation with various physical, clinical and metabolic parameters along with its inhibitory effect on NF-κB are suggestive of a protective role of HDAC4 against obesity. HDAC4 could therefore represent

  3. Integration of transcriptome, proteome and metabolism data reveals the alkaloids biosynthesis in Macleaya cordata and Macleaya microcarpa.

    Directory of Open Access Journals (Sweden)

    Jianguo Zeng

    Full Text Available BACKGROUND: The Macleaya spp., including Macleaya cordata and Macleaya microcarpa, are traditional anti-virus, inflammation eliminating, and insecticide herb medicines for their isoquinoline alkaloids. They are also known as the basis of the popular natural animal food addictive in Europe. However, few studies especially at genomics level were conducted on them. Hence, we performed the Macleaya spp. transcriptome and integrated it with iTRAQ proteome analysis in order to identify potential genes involved in alkaloids biosynthesis. METHODOLOGY AND PRINCIPAL FINDINGS: We elaborately designed the transcriptome, proteome and metabolism profiling for 10 samples of both species to explore their alkaloids biosynthesis. From the transcriptome data, we obtained 69367 and 78255 unigenes for M. cordata and M. microcarpa, in which about two thirds of them were similar to sequences in public databases. By metabolism profiling, reverse patterns for alkaloids sanguinarine, chelerythrine, protopine, and allocryptopine were observed in different organs of two species. We characterized the expressions of enzymes in alkaloid biosynthesis pathways. We also identified more than 1000 proteins from iTRAQ proteome data. Our results strongly suggest that the root maybe the organ for major alkaloids biosynthesis of Macleaya spp. Except for biosynthesis, the alkaloids storage and transport were also important for their accumulation. The ultrastructure of laticifers by SEM helps us to prove the alkaloids maybe accumulated in the mature roots. CONCLUSIONS/SIGNIFICANCE: To our knowledge this is the first study to elucidate the genetic makeup of Macleaya spp. This work provides clues to the identification of the potential modulate genes involved in alkaloids biosynthesis in Macleaya spp., and sheds light on researches for non-model medicinal plants by integrating different high-throughput technologies.

  4. Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain.

    Science.gov (United States)

    Taurino, Federica; Stanca, Eleonora; Siculella, Luisa; Trentadue, Raffaella; Papa, Sergio; Zanotti, Franco; Gnoni, Antonio

    2012-04-18

    Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.

  5. Comprehensive proteome quantification reveals NgBR as a new regulator for Epithelial-Mesenchymal Transition of breast tumor cells

    OpenAIRE

    Zhao, Baofeng; Xu, Bo; Hu, Wenquan; Song, Chunxia; Wang, Fangjun; Liu, Zhong; Ye, Mingliang; Zou, Hanfa; Miao, Qing R.

    2014-01-01

    Nogo-B receptor (NgBR) is a type I receptor and specifically binds to ligand Nogo-B. Our previous work has shown that NgBR is highly expressed in human breast invasive ductal carcinoma. Here, comprehensive proteome quantification was performed to examine the alteration of protein expression profile in MDA-MB-231 breast tumor cells after knocking down NgBR using lentivirus-mediated shRNA approach. Among a total of 1771 proteins feasibly quantified, 994 proteins were quantified in two biologica...

  6. Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L..

    Directory of Open Access Journals (Sweden)

    Shuzhen Wang

    Full Text Available 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

  7. Proteomic analysis of pycnogenol effects in RAW 264.7 macrophage reveals induction of cathepsin D expression and enhancement of phagocytosis.

    Science.gov (United States)

    Wu, Ting-Feng; Hsu, Chiung-Yueh; Huang, Huei-Sheng; Chou, Siao-Ping; Wu, Hung

    2007-11-28

    Pycnogenol, polyphenolic compounds extracted from the pine bark, is beneficial for human health. To understand more of its effects, the present study is to explore the protein expression pattern induced by pycnogenol in RAW 264.7 cells. Global analysis using two-dimensional gel electrophoresis indicated that treatment with pycnogenol induces upregulation of four proteins, whose identities were revealed by mass spectrometry as cathepsin D, keratinocyte lipid-binding protein, proteasome subunit alpha type 1, and annexin IV. The pycnogenol effect displayed a time- and concentration-dependent manner. Unlike pycnogenol, N-acetyl cysteine and vitamin C had no effect on cathepsin D expression. Further studies showed that cathepsin D induction is correlated with an increase of lysosomal staining and enhancement of phagocytosis. These results reveal the novel effects of pycnogenol on protein expression and phagocytic functions and illustrate the advantage of proteomics-based strategy in unveiling the molecular basis of phytochemicals.

  8. iTRAQ-based quantitative proteomics of stratum corneum of dandruff scalp reveals new insights into its aetiology and similarities with atopic dermatitis.

    Science.gov (United States)

    Cavusoglu, Nükhet; Delattre, Caroline; Donovan, Mark; Bourassa, Sylvie; Droit, Arnaud; El Rawadi, Charles; Jourdain, Roland; Bernard, Dominique

    2016-11-01

    The study aimed at detecting differentially expressed proteins in the stratum corneum of dandruff versus non-dandruff scalps to better understand dandruff aetiology. iTRAQ-based quantitative proteomic analysis revealed a total of 68 differentially expressed biomarkers. A detailed analysis of their known physiological functions provided new insights into the affected metabolic pathways of a dandruff scalp. Dandruff scalp showed (1) profound changes in the expression and maturation of structural and epidermal differentiation related proteins, that are responsible for the integrity of the skin, (2) altered relevant factors that regulate skin hydration, and (3) an imbalanced physiological protease-protease inhibitor ratio. Stratum corneum proteins with antimicrobial activity, mainly those derived from sweat and sebaceous glands were also found modified. Comparing our data with those reported for atopic dermatitis revealed that about 50 % of the differentially expressed proteins in the superficial layers of the stratum corneum from dandruff and atopic dermatitis are identical.

  9. Proteomic analysis of conidia germination in Fusarium oxysporum f. sp. cubense tropical race 4 reveals new targets in ergosterol biosynthesis pathway for controlling Fusarium wilt of banana.

    Science.gov (United States)

    Deng, Gui-Ming; Yang, Qiao-Song; He, Wei-Di; Li, Chun-Yu; Yang, Jing; Zuo, Cun-Wu; Gao, Jie; Sheng, Ou; Lu, Shao-Yun; Zhang, Sheng; Yi, Gan-Jun

    2015-09-01

    Conidial germination is a crucial step of the soilborne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a most important lethal disease of banana. In this study, a total of 3659 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic approach, of which 1009 were differentially expressed during conidial germination of the fungus at 0, 3, 7, and 11 h. Functional classification and bioinformatics analysis revealed that the majority of the differentially expressed proteins are involved in six metabolic pathways. Particularly, all differential proteins involved in the ergosterol biosynthesis pathway were significantly upregulated, indicating the importance of the ergosterol biosynthesis pathway to the conidial germination of Foc TR4. Quantitative RT-PCR, western blotting, and in vitro growth inhibition assay by several categories of fungicides on the Foc TR4 were used to validate the proteomics results. Four enzymes, C-24 sterol methyltransferase (ERG6), cytochrome P450 lanosterol C-14α-demethylase (EGR11), hydroxymethylglutaryl-CoA synthase (ERG13), and C-4 sterol methyl oxidase (ERG25), in the ergosterol biosynthesis pathway were identified and verified, and they hold great promise as new targets for effective inhibition of Foc TR4 early growth in controlling Fusarium wilt of banana. To the best of our knowledge, this report represents the first comprehensive study on proteomics profiling of conidia germination in Foc TR4. It provides new insights into a better understanding of the developmental processes of Foc TR4 spores. More importantly, by host plant-induced gene silencing (HIGS) technology, the new targets reported in this work allow us to develop novel transgenic banana leading to high protection from Fusarium wilt and to explore more effective antifungal drugs against either individual or multiple target proteins of Foc TR4.

  10. 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.

  11. Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.

    Directory of Open Access Journals (Sweden)

    Deborah L Diamond

    2010-01-01

    Full Text Available Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.

  12. Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.

    Science.gov (United States)

    Diamond, Deborah L; Syder, Andrew J; Jacobs, Jon M; Sorensen, Christina M; Walters, Kathie-Anne; Proll, Sean C; McDermott, Jason E; Gritsenko, Marina A; Zhang, Qibin; Zhao, Rui; Metz, Thomas O; Camp, David G; Waters, Katrina M; Smith, Richard D; Rice, Charles M; Katze, Michael G

    2010-01-01

    Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.

  13. Tissue-specific defense and thermo-adaptive mechanisms of soybean seedlings under heat stress revealed by proteomic approach.

    Science.gov (United States)

    Ahsan, Nagib; Donnart, Tifenn; Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2010-08-06

    A comparative proteomic approach was employed to explore tissue-specific protein expression patterns in soybean seedlings under heat stress. The changes in the protein expression profiles of soybean seedling leaves, stems, and roots were analyzed after exposure to high temperatures. A total of 54, 35, and 61 differentially expressed proteins were identified from heat-treated leaves, stems, and roots, respectively. Differentially expressed heat shock proteins (HSPs) and proteins involved in antioxidant defense were mostly up-regulated, whereas proteins associated with photosynthesis, secondary metabolism, and amino acid and protein biosynthesis were down-regulated in response to heat stress. A group of proteins, specifically low molecular weight HSPs and HSP70, were up-regulated and expressed in a similar manner in all tissues. Proteomic analysis indicated that the responses of HSP70, CPN-60 beta, and ChsHSP were tissue specific, and this observation was validated by immunoblot analysis. The heat-responsive sHSPs were not induced by other stresses such as cold and hydrogen peroxide. Taken together, these results suggest that to cope with heat stress soybean seedlings operate tissue-specific defenses and adaptive mechanisms, whereas a common defense mechanism associated with the induction of several HSPs was employed in all three tissues. In addition, tissue-specific proteins may play a crucial role in defending each type of tissues against thermal stress.

  14. Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation.

    Science.gov (United States)

    Szopinska, Aleksandra; Christ, Eva; Planchon, Sebastien; König, Helmut; Evers, Daniele; Renaut, Jenny

    2016-02-01

    During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially available S. cerevisiae strain. All strains examined showed (i) different fermentative behaviour, (ii) stress resistance as well as (iii) susceptibility to stuck fermentation which was reflected in significant differences in protein expression levels. During our research we identified differentially expressed proteins in 155 gel spots which correspond to 70 different protein functions. Differences of expression between strains were observed mainly among proteins involved in stress response, proteins degradation pathways, cell redox homeostasis and amino acids biosynthesis. Interestingly, the newly discovered triple hybrid S. cerevisiae × S. kudriavzevii × S. bayanus strain which has the ability to naturally restart stuck fermentation showed a very strong induction of expression of two proteolytic enzymes: Pep4 and Prc1 that appear as numerous isoforms on the gel image and which may be the key to its unique properties. This study is an important step towards the better understanding of wine fermentations at a molecular level.

  15. Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics

    Science.gov (United States)

    Jacobs, Jon M.; Sorensen, Christina M.; Walters, Kathie-Anne; Proll, Sean C.; McDermott, Jason E.; Gritsenko, Marina A.; Zhang, Qibin; Zhao, Rui; Metz, Thomas O.; Camp, David G.; Waters, Katrina M.; Smith, Richard D.; Rice, Charles M.; Katze, Michael G.

    2010-01-01

    Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis. PMID:20062526

  16. Comparative proteomics reveals that a saxitoxin-producing and a nontoxic strain of Anabaena circinalis are two different ecotypes.

    Science.gov (United States)

    D'Agostino, Paul M; Song, Xiaomin; Neilan, Brett A; Moffitt, Michelle C

    2014-03-01

    In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions

    Science.gov (United States)

    Vendelova, Emilia; Camargo de Lima, Jeferson; Lorenzatto, Karina Rodrigues; Monteiro, Karina Mariante; Mueller, Thomas; Veepaschit, Jyotishman; Grimm, Clemens; Brehm, Klaus; Hrčková, Gabriela; Lutz, Manfred B.; Ferreira, Henrique B.

    2016-01-01

    Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts. PMID:27736880

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. First proteome study of sporadic flowering in bamboo species (Bambusa vulgaris and Dendrocalamus manipureanus) reveal the boom is associated with stress and mobile genetic elements.

    Science.gov (United States)

    Louis, Bengyella; Waikhom, Sayanika Devi; Goyari, Sailendra; Jose, Robinson C; Roy, Pranab; Talukdar, Narayan Chandra

    2015-12-15

    Bamboo species are the fastest-growing plants having a long vegetative cycle. Abrupt switching from the vegetative phase to the reproductive phase via sporadic flowering boom, occasionally leads to death of bamboo clumps, and threatens the existence of many bamboo species. To apprehend the molecular mechanism driving sporadic flowering, proteome changes in the initial and advanced floral buds of two edible bamboo species (Bambusa vulgaris and Dendrocalamus manipureanus) was dissected by two-dimensional gel electrophoresis (2-DE). A total of 39 differentially expressed peptide spots were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF/MS). In both B. vulgaris and D. manipureanus, identified proteins were categorized as transposon-related, defence and stress-related, cell cycle related, metabolism related, signal transduction related, and some lacked known putative domains. Proteins such as SEPALLATA3, ubiquitin, histone 3, thaumatin-like protein, putative tethering factor, SF-assemblin, polyubiquitin, mitochondrial carrier-like protein and RPT2-like protein were significantly expressed. Differences in D. manipureanus and B. vulgaris suggested that bamboo species have diverse 'drivers' or 'passengers' genes that govern natural sporadic flowering boom. This first floral proteomics analysis of bamboos revealed that sporadic boom is a highly energetic process, associated with stress elements, mobile genetic elements and signal transduction cross-talk elements.

  2. 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.

  3. 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

  4. Extended multiplexing of TMT labeling reveals age and high fat diet specific proteome changes in mouse epididymal adipose tissue.

    Science.gov (United States)

    Plubell, Deanna L; Wilmarth, Phillip A; Zhao, Yuqi; Fenton, Alexandra M; Minnier, Jessica; Reddy, Ashok P; Klimek, John; Yang, Xia; David, Larry L; Pamir, Nathalie

    2017-03-21

    The lack of high-throughput methods to analyze the adipose tissue protein composition limits our understanding of the protein networks responsible for age and diet related metabolic response. We have developed an approach using multiple-dimension liquid chromatography tandem mass spectrometry and extended multiplexing (24 biological samples) with TMT labeling to analyze proteomes of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short or a long-term, and from mice that aged on low vs. high fat diets. The peripheral metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet and advancing age, with long-term high fat diet exposure being the worst. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g., AACS, ACOX1, ACLY) and red-ox pathways (e.g., CPD2, CYP2E, SOD3) were significantly altered (FDR fat diet significantly altered 55 proteins associated with immune response (e.g., IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g., OTC, ARG1, CPS1), and amino acid biosynthesis (e.g., GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g., ARG1, CPS1), immune response/complement activation (e.g., C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g., EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g., YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Using our adipose tissue tailored approach we have identified both age-related and high fat diet specific proteomic signatures highlighting a pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid

  5. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

    Directory of Open Access Journals (Sweden)

    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  6. 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...

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

    Science.gov (United States)

    Panda, Bandita; Basu, Bhakti; Acharya, Celin; Rajaram, Hema; Apte, Shree Kumar

    2017-01-01

    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 (LD50 dose), following 3h exposure to 75μM and 200μM 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.

  8. Proteomic analysis of mitochondria reveals a metabolic switch from fatty acid oxidation to glycolysis in the failing heart

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    This work characterizes the mitochondrial proteomic profile in the failing heart and elucidates the molecular basis of mitochondria in heart failure. Heart failure was induced in rats by myocardial infarction, and mitochondria were isolated from hearts by differential centrifugation. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a system biology approach was employed to investigate differences in mitochondrial proteins between normal and failing hearts. Mass spectrometry identified 27 proteins differentially expressed that involved in energy metabolism. Among those, the up-regulated proteins included tricarboxylic acid cycle enzymes and pyruvate dehydrogenase complex subunits while the down-regulated proteins were involved in fatty acid oxidation and the OXPHOS complex. These results suggest a substantial metabolic switch from free fatty acid oxidation to glycolysis in heart failure and provide molecular evidence for alterations in the structural and functional parameters of mitochondria that may contribute to cardiac dysfunction during ischemic injury.

  9. iTRAQ-Based Quantitative Proteomic Analysis of Cotton Roots and Leaves Reveals Pathways Associated with Salt Stress.

    Science.gov (United States)

    Chen, Tingting; Zhang, Lei; Shang, Haihong; Liu, Shaodong; Peng, Jun; Gong, Wankui; Shi, Yuzhen; Zhang, Siping; Li, Junwen; Gong, Juwu; Ge, Qun; Liu, Aiying; Ma, Huijuan; Zhao, Xinhua; Yuan, Youlu

    2016-01-01

    Salinity is a major abiotic stress that affects plant growth and development. In this study, we performed a proteomic analysis of cotton roots and leaf tissue following exposure to saline stress. 611 and 1477 proteins were differentially expressed in the roots and leaves, respectively. In the roots, 259 (42%) proteins were up-regulated and 352 (58%) were down-regulated. In the leaves, 748 (51%) proteins were up-regulated and 729 (49%) were down-regulated. On the basis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we concluded that the phenylalanine metabolism and starch and sucrose metabolism were active for energy homeostasis to cope with salt stress in cotton roots. Moreover, photosynthesis, pyruvate metabolism, glycolysis / gluconeogenesis, carbon fixation in photosynthetic organisms and phenylalanine metabolism were inhabited to reduce energy consumption. Characterization of the signaling pathways will help elucidate the mechanism activated by cotton in response to salt stress.

  10. Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.

    Science.gov (United States)

    Hewitson, James P; Ivens, Al C; Harcus, Yvonne; Filbey, Kara J; McSorley, Henry J; Murray, Janice; Bridgett, Stephen; Ashford, David; Dowle, Adam A; Maizels, Rick M

    2013-08-01

    Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H

  11. Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.

    Directory of Open Access Journals (Sweden)

    James P Hewitson

    2013-08-01

    Full Text Available Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4 larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL members, with acetylcholinesterases (ACEs and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel

  12. Proteomic and Lipidomic Analysis of Nanoparticle Corona upon Contact with Lung Surfactant Reveals Differences in Protein, but Not Lipid Composition.

    Science.gov (United States)

    Raesch, Simon Sebastian; Tenzer, Stefan; Storck, Wiebke; Rurainski, Alexander; Selzer, Dominik; Ruge, Christian Arnold; Perez-Gil, Jesus; Schaefer, Ulrich Friedrich; Lehr, Claus-Michael

    2015-12-22

    Pulmonary surfactant (PS) constitutes the first line of host defense in the deep lung. Because of its high content of phospholipids and surfactant specific proteins, the interaction of inhaled nanoparticles (NPs) with the pulmonary surfactant layer is likely to form a corona that is different to the one formed in plasma. Here we present a detailed lipidomic and proteomic analysis of NP corona formation using native porcine surfactant as a model. We analyzed the adsorbed biomolecules in the corona of three NP with different surface properties (PEG-, PLGA-, and Lipid-NP) after incubation with native porcine surfactant. Using label-free shotgun analysis for protein and LC-MS for lipid analysis, we quantitatively determined the corona composition. Our results show a conserved lipid composition in the coronas of all investigated NPs regardless of their surface properties, with only hydrophilic PEG-NPs adsorbing fewer lipids in total. In contrast, the analyzed NP displayed a marked difference in the protein corona, consisting of up to 417 different proteins. Among the proteins showing significant differences between the NP coronas, there was a striking prevalence of molecules with a notoriously high lipid and surface binding, such as, e.g., SP-A, SP-D, DMBT1. Our data indicate that the selective adsorption of proteins mediates the relatively similar lipid pattern in the coronas of different NPs. On the basis of our lipidomic and proteomic analysis, we provide a detailed set of quantitative data on the composition of the surfactant corona formed upon NP inhalation, which is unique and markedly different to the plasma corona.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. "Topological significance" analysis of gene expression and proteomic profiles from prostate cancer cells reveals key mechanisms of androgen response.

    Directory of Open Access Journals (Sweden)

    Adaikkalam Vellaichamy

    Full Text Available BACKGROUND: The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus. CONCLUSIONS/SIGNIFICANCE: We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an

  15. 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.

  16. Quantitative Proteomic and Transcriptomic Study on Autotetraploid Paulownia and Its Diploid Parent Reveal Key Metabolic Processes Associated with Paulownia Autotetraploidization.

    Science.gov (United States)

    Dong, Yanpeng; Deng, Minjie; Zhao, Zhenli; Fan, Guoqiang

    2016-01-01

    Polyploidy plays a very important role in speciation and plant evolution by way of genomic merging and doubling. In the process of polyploidy, rapid genomic, and transcriptomic changes have been observed and researched. However, proteomic divergence caused by the effects of polyploidization is still poorly understood. In the present study, we used iTRAQ coupled with mass spectrometry to quantitatively analyze proteomic changes in the leaves of autotetraploid Paulownia and its diploid parent. A total of 2963 proteins were identified and quantified. Among them, 463 differentially abundant proteins were detected between autotetraploid Paulownia and its diploid parent, and 198 proteins were found to be non-additively abundant in autotetraploid Paulownia, suggesting the presence of non-additive protein regulation during genomic merger and doubling. We also detected 1808 protein-encoding genes in previously published RNA sequencing data. We found that 59 of the genes that showed remarkable changes at mRNA level encoded proteins with consistant changes in their abundance levels, while a further 48 genes that showed noteworthy changes in their expression levels encoded proteins with opposite changes in their abundance levels. Proteins involved in posttranslational modification, protein turnover, and response to stimulus, were significantly enriched among the non-additive proteins, which may provide some of the driving power for variation and adaptation in autopolyploids. Quantitative real-time PCR analysis verified the expression patterns of related protein-coding genes. In addition, we found that the percentage of differentially abundant proteins that matched previously reported differentially expressed genes was relatively low.

  17. Interspecific proteomic comparisons reveal ash phloem genes potentially involved in constitutive resistance to the emerald ash borer.

    Directory of Open Access Journals (Sweden)

    Justin G A Whitehill

    Full Text Available 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.

  18. 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.

  19. Hydrogen peroxide acts on sensitive mitochondrial proteins to induce death of a fungal pathogen revealed by proteomic analysis.

    Directory of Open Access Journals (Sweden)

    Guozheng Qin

    Full Text Available How the host cells of plants and animals protect themselves against fungal invasion is a biologically interesting and economically important problem. Here we investigate the mechanistic process that leads to death of Penicillium expansum, a widespread phytopathogenic fungus, by identifying the cellular compounds affected by hydrogen peroxide (H(2O(2 that is frequently produced as a response of the host cells. We show that plasma membrane damage was not the main reason for H(2O(2-induced death of the fungal pathogen. Proteomic analysis of the changes of total cellular proteins in P. expansum showed that a large proportion of the differentially expressed proteins appeared to be of mitochondrial origin, implying that mitochondria may be involved in this process. We then performed mitochondrial sub-proteomic analysis to seek the H(2O(2-sensitive proteins in P. expansum. A set of mitochondrial proteins were identified, including respiratory chain complexes I and III, F(1F(0 ATP synthase, and mitochondrial phosphate carrier protein. The functions of several proteins were further investigated to determine their effects on the H(2O(2-induced fungal death. Through fluorescent co-localization and the use of specific inhibitor, we provide evidence that complex III of the mitochondrial respiratory chain contributes to ROS generation in fungal mitochondria under H(2O(2 stress. The undesirable accumulation of ROS caused oxidative damage of mitochondrial proteins and led to the collapse of mitochondrial membrane potential. Meanwhile, we demonstrate that ATP synthase is involved in the response of fungal pathogen to oxidative stress, because inhibition of ATP synthase by oligomycin decreases survival. Our data suggest that mitochondrial impairment due to functional alteration of oxidative stress-sensitive proteins is associated with fungal death caused by H(2O(2.

  20. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication...... initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  1. Isobaric Tags for Relative and Absolute Quantification-based Comparative Proteomics Reveals the Features of Plasma Membrane-Associated Proteomes of Pollen Grains and Pollen Tubes from Lilium davidii

    Institute of Scientific and Technical Information of China (English)

    Bing Han; Sixue Chen; Shaojun Dai; Ning Yang; Tai Wang

    2010-01-01

    Mature pollen grains (PGs) from most plant species are metabolically quiescent. However, once pollinated onto stigma, they quickly hydrate and germinate. APG can give rise to a vegetative cell-derived polarized pollen tube (PT), which represents a specialized polar cell. The polarized PT grows by the tip and requires interaction of different signaling molecules localized in the apical plasma membrane and active membrane trafficking. The mechanisms underlying the interaction and membrane trafficking are not well understood. In this work, we purified PG and PT plasma-membrane vesicles from Lilium davidii Duch. using the aqueous two-phase partition technique, then enriched plasma membrane proteins by using Brij58 and KCl to remove loosely bound contaminants. We identified 223 integral and membrane-associated proteins in the plasma membrane of PGs and PTs by using isobaric tags for relative and absolute quantification (iTRAQ) and 2-D high-performance liquid chromatography-tandem mass spectrometry. More than 68% of the proteins have putative transmembrane domains and/or lipid-modified motifs. Proteins involved in signal transduction, membrane trafficking and transport are predominant in the plasma-membrane proteome. We revealed most components of the clathrin-dependent endocytosis pathway. Statistical analysis revealed 14 proteins differentially expressed in the two development stages: in PTs, six upregulated and eight downregulated are mainly involved in signaling, transport and membrane trafficking. These results provide novel insights into polarized PT growth.

  2. Mass spectrometric proteomics reveals that nuclear protein positive cofactor PC4 selectively binds to cross-linked DNA by a trans-platinum anticancer complex.

    Science.gov (United States)

    Du, Zhifeng; Luo, Qun; Yang, Liping; Bing, Tao; Li, Xianchan; Guo, Wei; Wu, Kui; Zhao, Yao; Xiong, Shaoxiang; Shangguan, Dihua; Wang, Fuyi

    2014-02-26

    An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a trans-platinum anticancer complex, trans-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by trans-PtTz.

  3. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

    Science.gov (United States)

    Bell-Temin, Harris; Culver-Cochran, Ashley E; Chaput, Dale; Carlson, Christina M; Kuehl, Melanie; Burkhardt, Brant R; Bickford, Paula C; Liu, Bin; Stevens, Stanley M

    2015-12-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

  4. 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.

  5. Four conventional soybean [Glycine max (L.) Merrill] seeds exhibit different protein profiles as revealed by proteomic analysis.

    Science.gov (United States)

    Gomes, Luciana S; Senna, Raquel; Sandim, Vanessa; Silva-Neto, Mário A C; Perales, Jonas E A; Zingali, Russolina B; Soares, Márcia R; Fialho, Eliane

    2014-02-12

    Soybeans have several functional properties due to their composition and may exert beneficial health effects that are attributed to proteins and their derivative peptides. The present study aimed to analyze the protein profiles of four new conventional soybean seeds (BRS 257, BRS 258, BRS 267, and Embrapa 48) with the use of proteomic tools. Two-dimensional (2D) and one-dimensional (1D) gel electrophoreses were performed, followed by MALDI-TOF/TOF and ESI-Q-TOF mass spectrometry analyses, respectively. These two different experimental approaches allowed the identification of 117 proteins from 1D gels and 46 differentially expressed protein spots in 2D gels. BRS 267 showed the greatest diversity of identified spots in the 2D gel analyses. In the 1D gels, the major groups were storage (25-40%) and lipid metabolism (11-25%) proteins. The differences in protein composition between cultivars could indicate functional and nutritional differences and could direct the development of new cultivars.

  6. Involvement of potential pathways in malignant transformation from Oral Leukoplakia to Oral Squamous Cell Carcinoma revealed by proteomic analysis

    Directory of Open Access Journals (Sweden)

    Li Jing

    2009-08-01

    Full Text Available Abstract Background Oral squamous cell carcinoma (OSCC is one of the most common forms of cancer associated with the presence of precancerous oral leukoplakia. Given the poor prognosis associated with oral leukoplakia, and the difficulties in distinguishing it from cancer lesions, there is an urgent need to elucidate the molecular determinants and critical signal pathways underlying the malignant transformation of precancerous to cancerous tissue, and thus to identify novel diagnostic and therapeutic target. Results We have utilized two dimensional electrophoresis (2-DE followed by ESI-Q-TOF-LC-MS/MS to identify proteins differentially expressed in six pairs of oral leukoplakia tissues with dysplasia and oral squamous cancer tissues, each pair was collected from a single patient. Approximately 85 differentially and constantly expressed proteins (> two-fold change, P Conclusion Varying levels of differentially expressed proteins were possibly involved in the malignant transformation of oral leukoplakia. Their expression levels, bioprocess, and interaction networks were analyzed using a bioinformatics approach. This study shows that the three homologs of PA28 may play an important role in malignant transformation and is an example of a systematic biology study, in which functional proteomics were constructed to help to elucidate mechanistic aspects and potential involvement of proteins. Our results provide new insights into the pathogenesis of oral cancer. These differentially expressed proteins may have utility as useful candidate markers of OSCC.

  7. Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways.

    Science.gov (United States)

    Erdem, Cemal; Nagle, Alison M; Casa, Angelo J; Litzenburger, Beate C; Wang, Yu-Fen; Taylor, D Lansing; Lee, Adrian V; Lezon, Timothy R

    2016-09-01

    Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro.

  8. Proteomic Analysis of Human Brain Microvascular Endothelial Cells Reveals Differential Protein Expression in Response to Enterovirus 71 Infection

    Directory of Open Access Journals (Sweden)

    Wenying Luo

    2015-01-01

    Full Text Available 2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC, to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  9. Fusion-related host proteins are actively regulated by NA during influenza infection as revealed by quantitative proteomics analysis.

    Directory of Open Access Journals (Sweden)

    Zhiwei Sui

    Full Text Available Three recombinant influenza A viruses with different neuraminidases (NAs in the background of A/PR/8/34 (PR8, named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS and two-dimensional gel electrophoresis (2-DE, we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses.

  10. Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.)

    Science.gov (United States)

    Wang, Shuping; Zhang, Gaisheng; Zhang, Yingxin; Song, Qilu; Chen, Zheng; Wang, Junsheng; Guo, Jialin; Niu, Na; Wang, Junwei; Ma, Shoucai

    2015-01-01

    Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat. PMID:26136264

  11. Proteomic analysis of mitochondria reveals a metabolic switch from fatty acid oxidation to glycolysis in the failing heart

    Institute of Scientific and Technical Information of China (English)

    WANG Jun; WANG TingZhong; LIU Ping; FAN FenLing; GUAN YouFei; MA AiQun; BAI Ling; LI Jing; SUN ChaoFeng; ZHAO Jin; CUI ChangCong; HAN Ke; LIU Yu; ZHUO XiaoZhen

    2009-01-01

    This work characterizes the mitochondrial proteomic profile in the failing heart and elucidates the molecular basis of mitochondria in heart failure.Heart failure was induced in rats by myocardial infarction,and mitochondria were isolated from hearts by differential centrifugation.Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry,a system biology approach was employed to investigate differences in mitochondrial proteins between normal and failing hearts.Mass spectrometry identified 27 proteins differentially expressed that involved in energy metabolism.Among those,the up-regulated proteins included tricarboxylic acid cycle enzymes and pyruvate dehydrogenase complex subunita while the down-regulated proteins were involved in fatty acid oxidation and the OXPHOS complex.These results suggest a substantial metabolic switch from free fatty acid oxidation to glycolysis in heart failure and provide molecular evidence for alterations in the structural and functional parameters of mitochondria that may contribute to cardiac dysfunction during ischemic injury.

  12. 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.

  13. Quantitative proteomics reveals ecological fitness cost of multi-herbicide resistant barnyardgrass (Echinochloa crus-galli L.).

    Science.gov (United States)

    Yang, Xia; Zhang, Zichang; Gu, Tao; Dong, Mingchao; Peng, Qiong; Bai, Lianyang; Li, Yongfeng

    2017-01-06

    Barnyardgrass (Echinochloa crus-galli) is one of the top 15 herbicide-resistant weeds around the world that interferes with rice growth, resulting in major losses of rice yield. Thus, multi-herbicide resistance in barnyardgrass presents a major threat, with the underlying mechanisms that contribute to resistance requiring elucidation. In an attempt to characterize this multi-herbicide resistance at the proteomic level, comparative analysis of resistant and susceptible barnyardgrasses was performed using iTRAQ, both with and without quinclorac, bispyribac-sodium and penoxsulam herbicidal treatment. A total of 1342 protein species were identified from 2248 unique peptides by searching the UniProt database and conducting data analysis. Approximately 904 protein species with 4774 Gene Ontology (GO) terms were grouped into the categories of biological process, cellular component and molecular function. Among these, 688 protein species were annotated into 1583 KEGG pathways, with 980 protein species relating to metabolism and 93 relating to environmental information processing. A total of 292 protein species showed more than a 1.2-fold change in abundance in the resistant biotype relative to the susceptible biotype. Furthermore, herbicide treatment resulted in 157 protein species that showed more than a 1.2-fold change in the resistant biotype. Moreover, physiological analyses demonstrated an ecological fitness cost in the resistant biotype.

  14. Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity.

    Science.gov (United States)

    Tanou, Georgia; Job, Claudette; Rajjou, Loïc; Arc, Erwann; Belghazi, Maya; Diamantidis, Grigorios; Molassiotis, Athannasios; Job, Dominique

    2009-12-01

    Hydrogen peroxide (H(2)O(2)) and nitric oxide (*NO) are key reactive species in signal transduction pathways leading to activation of plant defense against biotic or abiotic stress. Here, we investigated the effect of pre-treating citrus plants (Citrus aurantium L.) with either of these two molecules on plant acclimation to salinity and show that both pre-treatments strongly reduced the detrimental phenotypical and physiological effects accompanying this stress. A proteomic analysis disclosed 85 leaf proteins that underwent significant quantitative variations in plants directly exposed to salt stress. A large part of these changes was not observed with salt-stressed plants pre-treated with either H(2)O(2) or sodium nitroprusside (SNP; a *NO-releasing chemical). We also identified several proteins undergoing changes either in their oxidation (carbonylation; 40 proteins) and/or S-nitrosylation (49 proteins) status in response to salinity stress. Both H(2)O(2) and SNP pre-treatments before salinity stress alleviated salinity-induced protein carbonylation and shifted the accumulation levels of leaf S-nitrosylated proteins to those of unstressed control plants. Altogether, the results indicate an overlap between H(2)O(2)- and *NO-signaling pathways in acclimation to salinity and suggest that the oxidation and S-nitrosylation patterns of leaf proteins are specific molecular signatures of citrus plant vigour under stressful conditions.

  15. Genomic and proteomic profiling reveals reduced mitochondrial function and disruption of the neuromuscular junction driving rat sarcopenia.

    Science.gov (United States)

    Ibebunjo, Chikwendu; Chick, Joel M; Kendall, Tracee; Eash, John K; Li, Christine; Zhang, Yunyu; Vickers, Chad; Wu, Zhidan; Clarke, Brian A; Shi, Jun; Cruz, Joseph; Fournier, Brigitte; Brachat, Sophie; Gutzwiller, Sabine; Ma, QiCheng; Markovits, Judit; Broome, Michelle; Steinkrauss, Michelle; Skuba, Elizabeth; Galarneau, Jean-Rene; Gygi, Steven P; Glass, David J

    2013-01-01

    Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia.

  16. 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.

  17. 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.

  18. Quantitative proteomic changes during post myocardial infarction remodeling reveals altered cardiac metabolism and Desmin aggregation in the infarct region.

    Science.gov (United States)

    Datta, Kaberi; Basak, Trayambak; Varshney, Swati; Sengupta, Shantanu; Sarkar, Sagartirtha

    2017-01-30

    Myocardial infarction is one of the leading causes of cardiac dysfunction, failure and sudden death. Post infarction cardiac remodeling presents a poor prognosis, with 30%-45% of patients developing heart failure, in a period of 5-25years. Oxidative stress has been labelled as the primary causative factor for cardiac damage during infarction, however, the impact it may have during the process of post infarction remodeling has not been well probed. In this study, we have implemented iTRAQ proteomics to catalogue proteins and functional processes, participating both temporally (early and late phases) and spatially (infarct and remote zones), during post myocardial infarction remodeling of the heart as functions of the differential oxidative stress manifest during the remodeling process. Cardiac metabolism was the dominant network to be affected during infarction and the remodeling time points considered in this study. A distinctive expression pattern of cytoskeletal proteins was also observed with increased remodeling time points. Further, it was found that the cytoskeletal protein Desmin, aggregated in the infarct zone during the remodeling process, mediated by the protease Calpain1. Taken together, all of these data in conjunction may lay the foundation to understand the effects of oxidative stress on the remodeling process and elaborate the mechanism behind the compromised cardiac function observed during post myocardial infarction remodeling.

  19. Proteomic analysis of α4β1 integrin adhesion complexes reveals α-subunit-dependent protein recruitment

    Science.gov (United States)

    Byron, Adam; Humphries, Jonathan D; Craig, Sue E; Knight, David; Humphries, Martin J

    2012-01-01

    Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events. PMID:22623428

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

    Directory of Open Access Journals (Sweden)

    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

  1. 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

  2. Reprogramming the chromatin landscape

    DEFF Research Database (Denmark)

    Miranda, Tina B; Voss, Ty C; Sung, Myong-Hee;

    2013-01-01

    , mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER...... and GR. Upon induction, GR modulates access of ER to specific sites in the genome by reorganization of the chromatin configuration for these elements. Binding to these newly accessible sites occurs either by direct recognition of ER response elements or indirectly through interactions with other factors...

  3. 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.

  4. Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways.

    Science.gov (United States)

    Govey, Peter M; Jacobs, Jon M; Tilton, Susan C; Loiselle, Alayna E; Zhang, Yue; Freeman, Willard M; Waters, Katrina M; Karin, Norman J; Donahue, Henry J

    2014-06-03

    Osteocytes, positioned within bone׳s porous structure, are subject to interstitial fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone adaptation to mechanical load. The objective of this study was to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels using high-throughput approaches. Osteocyte-like MLO-Y4 cells were subjected to 2h of oscillating fluid flow (1Pa peak shear stress) and analyzed following 0, 2, 8, and 24h post-flow incubation. Transcriptomic microarray analysis, followed by gene ontology pathway analysis, demonstrated fluid flow regulation of genes consistent with both known and unknown metabolic and inflammatory responses in bone. Additionally, two of the more highly up-regulated gene products - chemokines Cxcl1 and Cxcl2, supported by qPCR - have not previously been reported as responsive to fluid flow. Proteomic analysis demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. Finally, an integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including transcription factors c-Myc, c-Jun, and RelA/NF-κB. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as possible paracrine agents for osteoblastic and osteoclastic recruitment. Moreover, these results demonstrate the utility of integrative, high-throughput approaches in place of a traditional candidate approach for identifying novel mechano-sensitive signaling molecules.

  5. Proteome and Computational Analyses Reveal New Insights into the Mechanisms of Hepatitis C Virus Mediated Liver Disease Post-Transplantation

    Science.gov (United States)

    Diamond, Deborah L.; Krasnoselsky, Alexei L.; Burnum, Kristin E.; Monroe, Matthew E.; Webb-Robertson, Bobbie-Jo; McDermott, Jason E.; Yeh, Matthew M.; Dzib, Jose Felipe Golib; Susnow, Nathan; Strom, Susan; Proll, Sean C.; Belisle, Sarah E.; Purdy, David E.; Rasmussen, Angela L.; Walters, Kathie-Anne; Jacobs, Jon M.; Gritsenko, Marina A.; Camp, David G.; Bhattacharya, Renuka; Perkins, James D.; Carithers, Robert L.; Liou, Iris W.; Larson, Anne M.; Benecke, Arndt; Waters, Katrina M.; Smith, Richard D.; Katze, Michael G.

    2012-01-01

    Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV+ liver transplant recipients at 6 and/or 12 months post-transplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve (AUC). Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV+ liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in pro-inflammatory activity and impairment in anti-oxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. In conclusion, our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV+ liver

  6. Salmonella proteomics under oxidative stress reveals coordinated regulation of antioxidant defense with iron metabolism and bacterial virulence.

    Science.gov (United States)

    Fu, Jiaqi; Qi, Linlu; Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Qian; Liu, Xiaoyun

    2017-02-11

    Salmonella Typhimurium is a bacterial pathogen that can cause widespread gastroenteritis. Salmonella encounters reactive oxygen species both under free-living conditions and within their mammalian host during infection. To study its response to oxidative stress, we performed the first large-scale proteomic profiling of Salmonella upon exposure to H2O2. Among 1600 detected proteins, 83 proteins showed significantly altered abundance. Interestingly, only a subset of known antioxidants was induced, likely due to distinct regulatory mechanisms. In addition, we found elevation of several Salmonella acquired phage products with potential contribution to DNA repair under oxidative stress. Furthermore, we observed robust induction of iron-uptake systems and disruption of these pathways led to bacterial survival defects under H2O2 challenge. Importantly, this work is the first to report that oxidative stress severely repressed the Salmonella type III secretion system (T3SS), reducing its virulence. Biological significance Salmonella, a Gram-negative bacterial pathogen, encounters reactive oxygen species (ROS) both endogenously and exogenously. To better understand its response to oxidative stress, we performed the first large-scale profiling of Salmonella protein expression upon H2O2 treatment. Among 1600 quantified proteins, the abundance of 116 proteins was altered significantly. Notably, iron acquisition systems were induced to promote bacterial survival under oxidative stress. Furthermore, we are the first to report that oxidative stress severely repressed Salmonella type III secretion system and hence reduced its virulence. We believe that these findings will not only help us better understand the molecular mechanisms that Salmonella has evolved to counteract ROS but also the global impact of oxidative stress on bacterial physiology.

  7. Genome-wide transcriptomic and proteomic analyses of bollworm-infested developing cotton bolls revealed the genes and pathways involved in the insect pest defence mechanism.

    Science.gov (United States)

    Kumar, Saravanan; Kanakachari, Mogilicherla; Gurusamy, Dhandapani; Kumar, Krishan; Narayanasamy, Prabhakaran; Kethireddy Venkata, Padmalatha; Solanke, Amolkumar; Gamanagatti, Savita; Hiremath, Vamadevaiah; Katageri, Ishwarappa S; Leelavathi, Sadhu; Kumar, Polumetla Ananda; Reddy, Vanga Siva

    2016-06-01

    Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% of differentially expressed proteins were found to be involved in signalling followed by redox regulation. Further analysis showed that defence-related stress hormones and their lipid precursors, transcription factors, signalling molecules, etc. were stimulated, whereas the growth-related counterparts were suppressed during bollworm infestation. Around 26% of the significantly up-regulated proteins were defence molecules, while >50% of the significantly down-regulated were related to photosynthesis and growth. Interestingly, the biosynthesis genes for synergistically regulated jasmonate, ethylene and suppressors of the antagonistic factor salicylate were found to be up-regulated, suggesting a choice among stress-responsive phytohormone regulation. Manual curation of the enzymes and TFs highlighted the components of retrograde signalling pathways. Our data suggest that a selective regulatory mechanism directs the reallocation of metabolic resources favouring defence over growth under bollworm infestation and these insights could be exploited to develop bollworm-resistant cotton varieties.

  8. Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1.

    Science.gov (United States)

    Susor, Andrej; Ellederova, Zdenka; Jelinkova, Lucie; Halada, Petr; Kavan, Daniel; Kubelka, Michal; Kovarova, Hana

    2007-10-01

    In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.

  9. Proteomic analysis of chloroplast-to-chromoplast transition in tomato reveals metabolic shifts coupled with disrupted thylakoid biogenesis machinery and elevated energy-production components.

    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

    2012-10-01

    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.

  10. Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses

    Directory of Open Access Journals (Sweden)

    Brauer Aimee L

    2010-06-01

    Full Text Available Abstract Background Nontypeable Haemophilus influenzae colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, H. influenzae, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by H. influenzae in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient. Results A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions. Conclusions Proteomic expression profiling of H. influenzae grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that H. influenzae adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival

  11. CHD chromatin remodelers and the transcription cycle.

    Science.gov (United States)

    Murawska, Magdalena; Brehm, Alexander

    2011-01-01

    It is well established that ATP-dependent chromatin remodelers modulate DNA access of transcription factors and RNA polymerases by "opening" or "closing" chromatin structure. However, this view is far too simplistic. Recent findings have demonstrated that these enzymes not only set the stage for the transcription machinery to act but are actively involved at every step of the transcription process. As a consequence, they affect initiation, elongation, termination and RNA processing. In this review we will use the CHD family as a paradigm to illustrate the progress that has been made in revealing these new concepts.

  12. Proteomic profiling of the human T-cell nucleolus.

    Science.gov (United States)

    Jarboui, Mohamed Ali; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

    2011-12-01

    The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology.

  13. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja

    2012-01-01

    initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA...

  14. 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

  15. 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

  16. 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

  17. Chromatin dynamics in plants

    NARCIS (Netherlands)

    Fransz, P.F.; Jong, de J.H.

    2002-01-01

    Recent studies in yeast, animals and plants have provided major breakthroughs in unraveling the molecular mechanism of higher-order gene regulation. In conjunction with the DNA code, proteins that are involved in chromatin remodeling, histone modification and epigenetic imprinting form a large netwo

  18. Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150.

    Science.gov (United States)

    Elsworth, Brendan; Sanders, Paul R; Nebl, Thomas; Batinovic, Steven; Kalanon, Ming; Nie, Catherine Q; Charnaud, Sarah C; Bullen, Hayley E; de Koning Ward, Tania F; Tilley, Leann; Crabb, Brendan S; Gilson, Paul R

    2016-11-01

    The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.

  19. Comparative Proteomic and Physiological Analysis Reveals the Variation Mechanisms of Leaf Coloration and Carbon Fixation in a Xantha Mutant of Ginkgo biloba L.

    Directory of Open Access Journals (Sweden)

    Xinliang Liu

    2016-10-01

    Full Text Available Yellow-green leaf mutants are common in higher plants, and these non-lethal chlorophyll-deficient mutants are ideal materials for research on photosynthesis and plant development. A novel xantha mutant of Ginkgo biloba displaying yellow-colour leaves (YL and green-colour leaves (GL was identified in this study. The chlorophyll content of YL was remarkably lower than that in GL. The chloroplast ultrastructure revealed that YL had less dense thylakoid lamellae, a looser structure and fewer starch grains than GL. Analysis of the photosynthetic characteristics revealed that YL had decreased photosynthetic activity with significantly high nonphotochemical quenching. To explain these phenomena, we analysed the proteomic differences in leaves and chloroplasts between YL and GL of ginkgo using two-dimensional gel electrophoresis (2-DE coupled with MALDI-TOF/TOF MS. In total, 89 differential proteins were successfully identified, 82 of which were assigned functions in nine metabolic pathways and cellular processes. Among them, proteins involved in photosynthesis, carbon fixation in photosynthetic organisms, carbohydrate/energy metabolism, amino acid metabolism, and protein metabolism were greatly enriched, indicating a good correlation between differentially accumulated proteins and physiological changes in leaves. The identifications of these differentially accumulated proteins indicates the presence of a specific different metabolic network in YL and suggests that YL possess slower chloroplast development, weaker photosynthesis, and a less abundant energy supply than GL. These studies provide insights into the mechanism of molecular regulation of leaf colour variation in YL mutants.

  20. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    Science.gov (United States)

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-09-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency.

  1. Chromatin assembly using Drosophila systems.

    Science.gov (United States)

    Fyodorov, Dmitry V; Levenstein, Mark E

    2002-05-01

    To successfully study chromatin structure and activity in vitro, it is essential to have a chromatin assembly system that will prepare extended nucleosome arrays with highly defined protein content that resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. The Drosophila ATP-dependent chromatin assembly system described in this unit meets these requirements. The end product of the reaction described here has highly periodic extended arrays with physiologic spacing and positioning of the nucleosomes.

  2. Chromatin associations in Arabidopsis interphase nuclei

    Directory of Open Access Journals (Sweden)

    Veit eSchubert

    2014-11-01

    Full Text Available The arrangement of chromatin within interphase nuclei seems to be caused by topological constraints and related to gene expression depending on tissue and developmental stage. In yeast and animals it was found that homologous and heterologous chromatin association are required to realize faithful expression and DNA repair. To test whether such associations are present in plants we analysed Arabidopsis thaliana interphase nuclei by FISH using probes from different chromosomes. We found that chromatin fibre movement and variable associations, although in general relatively seldom, may occur between euchromatin segments along chromosomes, sometimes even over large distances. The combination of euchromatin segments bearing high or low co-expressing genes did not reveal different association frequencies probably due to adjacent genes of deviating expression patterns.Based on previous data and on FISH analyses presented here, we conclude that the global interphase chromatin organization in A. thaliana is relatively stable, due to the location of its ten centromeres at the nuclear periphery and of the telomeres mainly at the centrally localized nucleolus. Nevertheless, chromatin movement enables a flexible spatial genome arrangement in plant nuclei.

  3. NET23/STING promotes chromatin compaction from the nuclear envelope.

    Directory of Open Access Journals (Sweden)

    Poonam Malik

    Full Text Available Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173, strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

  4. Effects of fast neutrons on chromatin: dependence on chromatin structure

    Energy Technology Data Exchange (ETDEWEB)

    Radu, L. [Dept. of Molecular Genetics, V. Babes National Inst., Bd. Timisoara, Bucharest (Romania); Constantinescu, B. [Dept. of Cyclotron, H. Hulubei National Inst., Bucharest (Romania); Gazdaru, D. [Dept. of Biophysics, Physics Faculty, Univ. of Bucharest (Romania)

    2002-07-01

    The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin-proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)-heterochromatin (compact) ratio. (author)

  5. 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 funga

  6. Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

    Science.gov (United States)

    Parte, Priyanka P; Rao, Parimala; Redij, Shweta; Lobo, Vivian; D'Souza, Serena J; Gajbhiye, Rahul; Kulkarni, Vijay

    2012-10-22

    Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

  7. iTRAQ-based quantitative proteomic analysis reveals potential factors associated with the enhancement of phenazine-1-carboxamide production in Pseudomonas chlororaphis P3.

    Science.gov (United States)

    Jin, Xue-Jie; Peng, Hua-Song; Hu, Hong-Bo; Huang, Xian-Qing; Wang, Wei; Zhang, Xue-Hong

    2016-06-07

    Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

  8. Proteomic Analysis of Exosomes and Exosome-Free Conditioned Media From Human Osteosarcoma Cell Lines Reveals Secretion of Proteins Related to Tumor Progression.

    Science.gov (United States)

    Jerez, Sofía; Araya, Héctor; Thaler, Roman; Charlesworth, M Cristine; López-Solís, Remigio; Kalergis, Alexis M; Céspedes, Pablo F; Dudakovic, Amel; Stein, Gary S; van Wijnen, Andre J; Galindo, Mario

    2017-02-01

    Osteosarcomas are the most prevalent bone tumors in pediatric patients, but can also occur later in life. Bone tumors have the potential to metastasize to lung and occasionally other vital organs. To understand how osteosarcoma cells interact with their micro-environment to support bone tumor progression and metastasis, we analyzed secreted proteins and exosomes from three human osteosarcoma cell lines. Exosome isolation was validated by transmission electron microscopy (TEM) and immuno-blotting for characteristic biomarkers (CD63, CD9, and CD81). Exosomal and soluble proteins (less than 100 kDa) were identified by mass spectrometry analysis using nanoLC-MS/MS and classified by functional gene ontology clustering. We identified a secretome set of >3,000 proteins for both fractions, and detected proteins that are either common or unique among the three osteosarcoma cell lines. Protein ontology comparison of proteomes from exosomes and exosome-free fractions revealed differences in the enrichment of functional categories associated with different biological processes, including those related to tumor progression (i.e., angiogenesis, cell adhesion, and cell migration). The secretome characteristics of osteosarcoma cells are consistent with the pathological properties of tumor cells with metastatic potential. J. Cell. Biochem. 118: 351-360, 2017. © 2016 Wiley Periodicals, Inc.

  9. Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.

    Directory of Open Access Journals (Sweden)

    Liping Ren

    Full Text Available Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

  10. 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.

  11. 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. PMID:28134344

  12. Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

    Science.gov (United States)

    Fazakerley, Daniel J; Naghiloo, Sheyda; Chaudhuri, Rima; Koumanov, Françoise; Burchfield, James G; Thomas, Kristen C; Krycer, James R; Prior, Matthew J; Parker, Ben L; Murrow, Beverley A; Stöckli, Jacqueline; Meoli, Christopher C; Holman, Geoffrey D; James, David E

    2015-09-25

    Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

  13. 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.

  14. Metabolomic and proteomic profiles reveal the dynamics of primary metabolism during seed development of lotus (Nelumbo nucifera

    Directory of Open Access Journals (Sweden)

    Pingfang eYang

    2016-06-01

    Full Text Available Sacred lotus (Nelumbo nucifera belongs to Nelumbonaceae family. Its seeds are widely consumed in Asia countries as snacks or even medicine. Besides the market values, lotus seed also plays crucial roles in lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP which was corresponding to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal the metabolism adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acids metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide reference data set for the evaluation of primary metabolism during lotus seed development.

  15. Metabolomic and Proteomic Profiles Reveal the Dynamics of Primary Metabolism during Seed Development of Lotus (Nelumbo nucifera).

    Science.gov (United States)

    Wang, Lei; Fu, Jinlei; Li, Ming; Fragner, Lena; Weckwerth, Wolfram; Yang, Pingfang

    2016-01-01

    Sacred lotus (Nelumbo nucifera) belongs to the Nelumbonaceae family. Its seeds are widely consumed in Asian countries as snacks or even medicine. Besides the market value, lotus seed also plays a crucial role in the lotus life cycle. Consequently, it is essential to gain a comprehensive understanding of the development of lotus seed. During its development, lotus seed undergoes cell division, expansion, reserve accumulation, desiccation, and maturation phases. We observed morphological and biochemical changes from 10 to 25 days after pollination (DAP) which corresponded to the reserve synthesis and accumulation phase. The volume of the seed expanded until 20 DAP with the color of the seed coat changing from yellow-green to dark green and gradually fading again. Starch and protein rapidly accumulated from 15 to 20 DAP. To further reveal metabolic adaptation, primary metabolites and proteins profiles were obtained using mass spectrometry based platforms. Metabolites and enzymes involved in sugar metabolism, glycolysis, TCA cycle and amino acid metabolism showed sequential dynamics enabling the clear separation of the different metabolic states during lotus seed development. The integration of the data revealed a highly significant metabolic switch at 15 DAP going through a transition of metabolically highly active tissue to the preparation of storage tissue. The results provide a reference data set for the evaluation of primary metabolism during lotus seed development.

  16. Global chromatin fibre compaction in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Charlotte [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Hayward, Richard L. [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Gilbert, Nick, E-mail: Nick.Gilbert@ed.ac.uk [Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom); Breakthrough Research Unit, The University of Edinburgh, Edinburgh EH4 2XR (United Kingdom)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  17. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Khotin, Mikhail, E-mail: h_mg@mail.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Turoverova, Lidia [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Aksenova, Vasilisa [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Barlev, Nikolai [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom); Borutinskaite, Veronika Viktorija [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius (Lithuania); Vener, Alexander [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Bajenova, Olga [Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Magnusson, Karl-Eric [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Pinaev, George P. [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Tentler, Dmitri, E-mail: dtentler@mail.cytspb.rssi.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation)

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  18. Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival.

    Science.gov (United States)

    Smithson, Laura J; Gutmann, David H

    2016-06-15

    As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Herein, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for mTOR-mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types.

  19. 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.

  20. Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain

    Directory of Open Access Journals (Sweden)

    Hare Nathan J

    2012-01-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF. While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. Results A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute isolate of the Australian epidemic strain 1 (AES-1R, the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. Conclusions Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients.

  1. A Broad Set of Chromatin Factors Influences Splicing

    Science.gov (United States)

    Allemand, Eric; Myers, Michael P.; Garcia-Bernardo, Jose; Harel-Bellan, Annick; Krainer, Adrian R.; Muchardt, Christian

    2016-01-01

    Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing. PMID:27662573

  2. Effect of Pre-Stressing on the Acid-Stress Response in Bifidobacterium Revealed Using Proteomic and Physiological Approaches.

    Directory of Open Access Journals (Sweden)

    Junhua Jin

    Full Text Available Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR, caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR, and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH3, thiols, and peptidoglycan, the activity of H+-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing.

  3. Proteomics in epigenetics: new perspectives for cancer research.

    Science.gov (United States)

    Bartke, Till; Borgel, Julie; DiMaggio, Peter A

    2013-05-01

    The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation. Over the recent years, mass spectrometry (MS) has become an indispensable tool in epigenetics research. In this review, we will give an overview of the applications of MS-based proteomics in studying various aspects of chromatin biology. We will focus on the use of MS in the discovery and mapping of histone modifications and how novel proteomic approaches are being utilized to identify and study chromatin-associated proteins and multi-subunit complexes. Finally, we will discuss the application of proteomic methods in the diagnosis and prognosis of cancer based on epigenetic biomarkers and comment on their future impact on cancer epigenetics.

  4. Cas9 Functionally Opens Chromatin.

    Directory of Open Access Journals (Sweden)

    Amira A Barkal

    Full Text Available Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding.

  5. 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.

  6. A label-free differential proteomics analysis reveals the effect of melatonin on promoting fruit ripening and anthocyanin accumulation upon postharvest in tomato.

    Science.gov (United States)

    Sun, Qianqian; Zhang, Na; Wang, Jinfang; Cao, Yunyun; Li, Xingsheng; Zhang, Haijun; Zhang, Lei; Tan, Dun-Xian; Guo, Yang-Dong

    2016-09-01

    To better understand the function of melatonin in tomato fruit ripening and quality improvement, a label-free quantitation method was used to investigate the proteins that differ between the control (CK) and 50 μm melatonin treatment (M50) fruits. Proteomics data identified 241 proteins that were significantly influenced by melatonin. These proteins were involved in several ripening-related pathways, including cell wall metabolism, oxidative phosphorylation, carbohydrate, and fatty acid metabolism. Moreover, the application of exogenous melatonin increased eight proteins that are related to anthocyanin accumulation during fruit ripening. Additionally, the affected protein levels correlated with the corresponding gene transcript levels. Further, the total anthocyanin content from M50 increased by 52%, 48%, and 50% at 5, 8, and 13 DAT (day after melatonin treatment), respectively. The melatonin-mediated promotion of fruit ripening and quality might be due to the altered proteins involved in processes associated with ripening. In this work, we indicated that a senescence-related protein was downregulated in the M50 fruit, while a cell apoptosis inhibitor (API5) protein was upregulated. In addition, peroxidases (POD9, POD12, peroxidase p7-like) and catalase (CAT3) significantly increased in the M50 fruits. Based on the previous studies and our data, we inferred that melatonin might be positively related to fruit ripening but negatively related to fruit senescence. This research provides insights into the physiological and molecular mechanisms underlying melatonin-mediated fruit ripening as well as the anthocyanin formation process in tomato fruit at the protein concentration level, and we reveal possible candidates for regulation of anthocyanin formation during fruit ripening.

  7. 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.

  8. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells.

    Science.gov (United States)

    Surmann, Kristin; Michalik, Stephan; Hildebrandt, Petra; Gierok, Philipp; Depke, Maren; Brinkmann, Lars; Bernhardt, Jörg; Salazar, Manuela G; Sun, Zhi; Shteynberg, David; Kusebauch, Ulrike; Moritz, Robert L; Wollscheid, Bernd; Lalk, Michael; Völker, Uwe; Schmidt, Frank

    2014-01-01

    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 2 × 10(6) bacteria, roughly 1450 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 decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins 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 regulatory

  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. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

    Science.gov (United States)

    Surmann, Kristin; Michalik, Stephan; Hildebrandt, Petra; Gierok, Philipp; Depke, Maren; Brinkmann, Lars; Bernhardt, Jörg; Salazar, Manuela G.; Sun, Zhi; Shteynberg, David; Kusebauch, Ulrike; Moritz, Robert L.; Wollscheid, Bernd; Lalk, Michael; Völker, Uwe; Schmidt, Frank

    2014-01-01

    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 2 × 106 bacteria, roughly 1450 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 decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins 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 regulatory

  11. Lung neuroendocrine tumours: deep sequencing of the four World Health Organization histotypes reveals chromatin-remodelling genes as major players and a prognostic role for TERT, RB1, MEN1 and KMT2D.

    Science.gov (United States)

    Simbolo, Michele; Mafficini, Andrea; Sikora, Katarzyna O; Fassan, Matteo; Barbi, Stefano; Corbo, Vincenzo; Mastracci, Luca; Rusev, Borislav; Grillo, Federica; Vicentini, Caterina; Ferrara, Roberto; Pilotto, Sara; Davini, Federico; Pelosi, Giuseppe; Lawlor, Rita T; Chilosi, Marco; Tortora, Giampaolo; Bria, Emilio; Fontanini, Gabriella; Volante, Marco; Scarpa, Aldo

    2017-03-01

    Next-generation sequencing (NGS) was applied to 148 lung neuroendocrine tumours (LNETs) comprising the four World Health Organization classification categories: 53 typical carcinoid (TCs), 35 atypical carcinoid (ACs), 27 large-cell neuroendocrine carcinomas, and 33 small-cell lung carcinomas. A discovery screen was conducted on 46 samples by the use of whole-exome sequencing and high-coverage targeted sequencing of 418 genes. Eighty-eight recurrently mutated genes from both the discovery screen and current literature were verified in the 46 cases of the discovery screen, and validated on additional 102 LNETs by targeted NGS; their prevalence was then evaluated on the whole series. Thirteen of these 88 genes were also evaluated for copy number alterations (CNAs). Carcinoids and carcinomas shared most of the altered genes but with different prevalence rates. When mutations and copy number changes were combined, MEN1 alterations were almost exclusive to carcinoids, whereas alterations of TP53 and RB1 cell cycle regulation genes and PI3K/AKT/mTOR pathway genes were significantly enriched in carcinomas. Conversely, mutations in chromatin-remodelling genes, including those encoding histone modifiers and members of SWI-SNF complexes, were found at similar rates in carcinoids (45.5%) and carcinomas (55.0%), suggesting a major role in LNET pathogenesis. One AC and one TC showed a hypermutated profile associated with a POLQ damaging mutation. There were fewer CNAs in carcinoids than in carcinomas; however ACs showed a hybrid pattern, whereby gains of TERT, SDHA, RICTOR, PIK3CA, MYCL and SRC were found at rates similar to those in carcinomas, whereas the MEN1 loss rate mirrored that of TCs. Multivariate survival analysis revealed RB1 mutation (p = 0.0005) and TERT copy gain (p = 0.016) as independent predictors of poorer prognosis. MEN1 mutation was associated with poor prognosis in AC (p = 0.0045), whereas KMT2D mutation correlated with longer survival in SCLC

  12. 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.

  13. Chromatin Flavors: Chromatin composition and domain organization in Drosophila melanogaster

    NARCIS (Netherlands)

    J.G. van Bemmel (Joke)

    2012-01-01

    textabstractChromatin was originally identified by W. Flemming in 1882 as not much more than the stainable substance of the cell nucleus. Flemming named this substance according to the Greek word “chroma”, meaning color. In 1911 chromatin was characterized as proteins, named histones, that were atta

  14. 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

    Intestinal ischemia and reperfusion injury are widely used models, which result into tissue injury and multiple organ failure also observed after trauma and surgery. Ischemic preconditioning (IPC) preceding ischemia and reperfusion (IR) was shown to attenuate this injury and has a potential...... 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...... is unclear. Proteomic analysis has been ratified as an appropriate tool for studying complex systems. In order to evaluate the effect of IPC preceding 45min of ischemia on the proteome of neutrophils we used Wistar rats divided in four experimental groups: Control, sham laparotomy, intestinal ischemia...

  15. Epigenetics & chromatin: Interactions and processes

    NARCIS (Netherlands)

    S. Henikoff (Steven); F.G. Grosveld (Frank)

    2013-01-01

    textabstractOn 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the confer

  16. The seed nuclear proteome.

    Science.gov (United States)

    Repetto, Ombretta; Rogniaux, Hélène; Larré, Colette; Thompson, Richard; Gallardo, Karine

    2012-01-01

    Understanding the regulatory networks coordinating seed development will help to manipulate seed traits, such as protein content and seed weight, in order to increase yield and seed nutritional value of important food crops, such as legumes. Because of the cardinal role of the nucleus in gene expression, sub-proteome analyses of nuclei from developing seeds were conducted, taking advantage of the sequences available for model species. In this review, we discuss the strategies used to separate and identify the nuclear proteins at a stage when the seed is preparing for reserve accumulation. We present how these data provide an insight into the complexity and distinctive features of the seed nuclear proteome. We discuss the presence of chromatin-modifying enzymes and proteins that have roles in RNA-directed DNA methylation and which may be involved in modifying genome architecture in preparation for seed filling. Specific features of the seed nuclei at the transition between the stage of cell divisions and that of cell expansion and reserve deposition are described here which may help to manipulate seed quality traits, such as seed weight.

  17. SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Treffers, Louise W; Verlaan-de Vries, Matty

    2015-01-01

    with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified......Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment...... dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors, and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1, and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO...

  18. Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

    Science.gov (United States)

    Lardenois, Aurélie; Jagot, Sabrina; Lagarrigue, Mélanie; Guével, Blandine; Ledevin, Mireille; Larcher, Thibaut; Dubreil, Laurence; Pineau, Charles; Rouger, Karl; Guével, Laëtitia

    2016-07-01

    Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

  19. A Proteomic Analysis of the Chromoplasts Isolated from Sweet Orange Fruits [Citrus sinensis (L.) Osbeck] Revealed Clues to Their Ripening Event

    Institute of Scientific and Technical Information of China (English)

    Yunliu Zeng; Zhiyong Pan; Yuduan Ding; Andan Zhu; Hongbo Cao; Qiang Xu; Xiuxin Deng

    2012-01-01

    Carotenoid biosynthesisis believed to occur in chromoplasts,which are non-photosynthetic plastids often present in flowers and fruits.We report a comprehensive proteomic analysis of the chromoplasts,which were purified from sweet orange by a novel protocol using Nycodenz density gradient centrifugation.Western blot analysis using polyclonal antibodies against marker proteins for different cell compartments suggested the chromoplast extraction were of high purity.Systematic analysis of the chromoplast proteome identified 493 proteins,of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses.Based on the predicted functions of these identified plastid proteins,a large proportion (~60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism,amino acid/protein synthesis,and secondary metabolism.Of note,HDS (hydroxymethylbutenyl 4-diphosphate synthase),PAP (plastid-lipid-associated protein),and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified.A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways.However,the citrus chromoplast was characterized by more extensive carotenoid synthesis,extensive amino acid synthesis without nitrogen assimilation,and evidence for lipid metabolism concerning jasmonic acid synthesis.In conclusion,this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

  20. New pleiotropic effects of eliminating a rare tRNA from Streptomyces coelicolor, revealed by combined proteomic and transcriptomic analysis of liquid cultures

    Directory of Open Access Journals (Sweden)

    Hotchkiss Graham

    2007-08-01

    Full Text Available Abstract Background In Streptomyces coelicolor, bldA encodes the only tRNA for a rare leucine codon, UUA. This tRNA is unnecessary for growth, but is required for some aspects of secondary metabolism and morphological development. We describe a transcriptomic and proteomic analysis of the effects of deleting bldA on cellular processes during submerged culture: conditions relevant to the industrial production of antibiotics. Results At the end of rapid growth, a co-ordinated transient up-regulation of about 100 genes, including many for ribosomal proteins, was seen in the parent strain but not the ΔbldA mutant. Increased basal levels of the signal molecule ppGpp in the mutant strain may be responsible for this difference. Transcripts or proteins from a further 147 genes classified as bldA-influenced were mostly expressed late in culture in the wild-type, though others were significantly transcribed during exponential growth. Some were involved in the biosynthesis of seven secondary metabolites; and some have probable roles in reorganising metabolism after rapid growth. Many of the 147 genes were "function unknown", and may represent unknown aspects of Streptomyces biology. Only two of the 147 genes contain a TTA codon, but some effects of bldA could be traced to TTA codons in regulatory genes or polycistronic operons. Several proteins were affected post-translationally by the bldA deletion. There was a statistically significant but weak positive global correlation between transcript and corresponding protein levels. Different technical limitations of the two approaches were a major cause of discrepancies in the results obtained with them. Conclusion Although deletion of bldA has very conspicuous effects on the gross phenotype, the bldA molecular phenotype revealed by the "dualomic" approach has shown that only about 2% of the genome is affected; but this includes many previously unknown effects at a variety of different levels, including post

  1. Comparative proteomic analysis using 2DE-LC-MS/MS reveals the mechanism of Fuzhuan brick tea extract against hepatic fat accumulation in rats with nonalcoholic fatty liver disease.

    Science.gov (United States)

    Liu, Zhonghua; Lin, Yong; Zhang, Sheng; Wang, Die; Liang, Qionglin; Luo, Guoan

    2015-09-01

    Fuzhuan brick tea has received increasing attention in recent years owing to its benefits for nonalcoholic fatty liver disease (NAFLD) and associated metabolic syndrome. For exploring the ameliorative mechanism, the liver proteomes from three groups of rats fed either a normal control diet (NCD), a high fat diet (HFD), or a HFD supplemented with high-dose Fuzhuan brick tea extract (FTE) (HFD + HFTE) were comprehensively compared by quantitative proteomics using 2DE-LC-MS/MS. This is the first study of the effects of tea aqueous extract on the liver proteome of rats suffering from metabolic syndrome. The results showed that 57 proteins displayed more than 1.5-fold differences in at least one of two comparisons of HFD versus NCD and HFD versus HFD + HFTE due to HFD feeding and FTE treatment, respectively. Of them, over 75% of proteins exhibited a similar tendency of expression in the two comparisons, meaning FTE was able to correct HFD effects on rat livers. By function analyses, an extensive list of proteins was involved in sugar and lipid metabolism. Compared with HFD-fed rats, the reduced lipogenesis and enhanced β-oxidation, tricarboxylic acid cycle and respiratory chain in HFD + HFTE-fed rats, which mainly contributed to ameliorate hepatic fat accumulation and associated NAFLD. Additionally, some putative drug targets were also revealed such as COX2, PGAM1, ACACB, FAS, and ECHS1.

  2. Nucleosome positioning and composition modulate in silico chromatin flexibility.

    Science.gov (United States)

    Clauvelin, N; Lo, P; Kulaeva, O I; Nizovtseva, E V; Diaz-Montes, J; Zola, J; Parashar, M; Studitsky, V M; Olson, W K

    2015-02-18

    The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of ∼150 DNA base pairs and eight histone proteins-found on chromatin fibers. Here we introduce a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs. We explore the effects of nucleosome positioning and the presence or absence of cationic N-terminal histone tails on the 'local' inter-nucleosomal interactions and the global deformations of the simulated chains. The correspondence between the predicted and observed effects of nucleosome composition and numbers on the long-range communication between the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We also extract effective nucleosome-nucleosome potentials from the simulations and implement the potentials in a larger-scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable effect of nucleosome spacing on chromatin flexibility, with small changes in DNA linker length significantly altering the interactions of nucleosomes and the dimensions of the fiber as a whole. In addition, we find that these changes in nucleosome positioning influence the statistical properties of long chromatin constructs. That is, simulated chromatin fibers with the same number of nucleosomes exhibit polymeric behaviors ranging from Gaussian to worm-like, depending upon nucleosome spacing. These findings suggest that the physical and mechanical properties of chromatin can span a wide range of behaviors, depending on nucleosome positioning, and

  3. Structure of chromatin in spermatozoa.

    Science.gov (United States)

    Björndahl, Lars; Kvist, Ulrik

    2014-01-01

    The specialized structure of the sperm chromatin has a dual function - first to protect the DNA from damage during storage and transport to the oocyte, and then to enable a rapid and complete unpacking of the undamaged paternal genome in the ooplasm. It is evident that zinc has a pivotal role in maintaining the structural stability and in enabling a rapid decondensation at the appropriate time. It is important for the sperm chromatin structure that the spermatozoa are ejaculated together with the zinc-rich prostatic secretion. Early exposure to zinc-binding seminal vesicular fluid can deplete the sperm chromatin of zinc and most likely induce surplus formation of disulfide bridges, likely to cause incomplete and delayed decondensation of the sperm chromatin in the oocyte. A premature decrease in sperm chromatin structure stability is likely to increase the risk for damage to the DNA due to increased access to the genome for DNA damaging compounds. The status of the sperm chromatin structure can vary in vitro depending on the exposure to zinc-depleting conditions when spermatozoa are stored in semen after ejaculation. When sperm DNA damage tests are evaluated and validated, it is therefore essential to also take into account the dynamics of zinc-dependent and zinc-independent sperm chromatin stability.

  4. R-loop: an emerging regulator of chromatin dynamics

    Institute of Scientific and Technical Information of China (English)

    Qais Al-Hadid; Yanzhong Yang

    2016-01-01

    The dynamic structure of chromatin,which exists in two conformational states:heterochromatin and euchromatin,alters the accessibility of the DNA to regulatory factors during transcription,replication,recombination,and DNA damage repair.Chemical modifications of histones and DNA,as well as adenosine triphospahate-dependent nucleosome remodeling,have been the major focus of research on chromatin dynamics over the past two decades.However,recent studies using a DNA-RNA hybrid-specific antibody and next-generation seque,ncing approaches have revealed that the formation of R-loops,one of the most common non-canonical DNA structures,is an emerging regulator of chromatin states.This review focuses on recent insights into the interplay between R-loop formation and the epigenetic modifications of chromatin in normal and disease states.

  5. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  6. Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum.

    Science.gov (United States)

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Singh, Vinay; Singh, Harikesh Bahadur

    2015-06-15

    Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants.

  7. Computational Biology in Clinical Proteomics and Chromatin Genomics

    NARCIS (Netherlands)

    Meuleman, W.

    2012-01-01

    The work in this thesis is concerned with two very distinct biological fields. The first part pertains to the development of techniques to aid in the search for clinical biomarkers for use in the early detection of cancer. The second part aims to elucidate in what way a genome is organised in a cell

  8. Genome‐wide transcriptomic and proteomic analyses of bollworm‐infested developing cotton bolls revealed the genes and pathways involved in the insect pest defence mechanism

    OpenAIRE

    Kumar, Saravanan; Kanakachari, Mogilicherla; Gurusamy, Dhandapani; Kumar, Krishan; Narayanasamy, Prabhakaran; Kethireddy Venkata, Padmalatha; Solanke, Amolkumar; Gamanagatti, Savita; Hiremath, Vamadevaiah; Katageri, Ishwarappa S.; Leelavathi, Sadhu; Kumar, Polumetla Ananda; Reddy, Vanga Siva

    2016-01-01

    Summary Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome‐wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi‐quantitative real‐time PCR. Comparative ana...

  9. Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation.

    Science.gov (United States)

    Ji, Yanlong; Wei, Shasha; Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

    2017-01-06

    Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

  10. Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms.

    Science.gov (United States)

    Bocchinfuso, Donald G; Taylor, Paul; Ross, Eric; Ignatchenko, Alex; Ignatchenko, Vladimir; Kislinger, Thomas; Pearson, Bret J; Moran, Michael F

    2012-09-01

    The freshwater planarian Schmidtea mediterranea has been used in research for over 100 years, and is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. Exteriorly, planarians are covered in mucous secretions of unknown composition, implicated in locomotion, predation, innate immunity, and substrate adhesion. Although the planarian genome has been sequenced, it remains mostly unannotated, challenging both genomic and proteomic analyses. The goal of the current study was to annotate the proteome of the whole planarian and its mucous fraction. The S. mediterranea proteome was analyzed via mass spectrometry by using multidimensional protein identification technology with whole-worm tryptic digests. By using a proteogenomics approach, MS data were searched against an in silico translated planarian transcript database, and by using the Swiss-Prot BLAST algorithm to identify proteins similar to planarian queries. A total of 1604 proteins were identified. The mucous subproteome was defined through analysis of a mucous trail fraction and an extract obtained by treating whole worms with the mucolytic agent N-acetylcysteine. Gene Ontology analysis confirmed that the mucous fractions were enriched with secreted proteins. The S. mediterranea proteome is highly similar to that predicted for the trematode Schistosoma mansoni associated with intestinal schistosomiasis, with the mucous subproteome particularly highly conserved. Remarkably, orthologs of 119 planarian mucous proteins are present in human mucosal secretions and tear fluid. We suggest planarians have potential to be a model system for the characterization of mucous protein function and relevant to parasitic flatworm infections and diseases underlined by mucous aberrancies, such as cystic fibrosis, asthma, and other lung diseases.

  11. Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Guanhui [CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing (China); University of Chinese Academy of Sciences, Beijing (China); Dong, Hongjun; Zhu, Yan; Mao, Shaoming [CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing (China); Zhang, Tianrui [CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing (China); Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin (China); Zhang, Yanping [CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing (China); Chen, Zugen [Department of Human Genetics, School of Medicine, University of California, Los Angeles, CA 90095 (United States); Li, Yin, E-mail: yli@im.ac.cn [CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing (China)

    2014-08-08

    Highlights: • Genomes of a butanol tolerant strain and its parent strain were deciphered. • Comparative genomic and proteomic was applied to understand butanol tolerance. • None differentially expressed proteins have mutations in its corresponding genes. • Mutations in ribosome might be responsible for the global difference of proteomics. - Abstract: Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19 g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work, we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.

  12. Vernalization-mediated chromatin changes.

    Science.gov (United States)

    Zografos, Brett R; Sung, Sibum

    2012-07-01

    Proper flowering time is vital for reproductive fitness in flowering plants. In Arabidopsis, vernalization is mediated primarily through the repression of a MADS box transcription factor, FLOWERING LOCUS C (FLC). The induction of a plant homeodomain-containing protein, VERNALIZATION INSENSITIVE 3 (VIN3), by vernalizing cold is required for proper repression of FLC. One of a myriad of changes that occurs after VIN3 is induced is the establishment of FLC chromatin at a mitotically repressed state due to the enrichment of repressive histone modifications. VIN3 induction by cold is the earliest known event during the vernalization response and includes changes in histone modifications at its chromatin. Here, the current understanding of the vernalization-mediated chromatin changes in Arabidopsis is discussed, with a focus on the roles of shared chromatin-modifying machineries in regulating VIN3 and FLC gene family expression during the course of vernalization.

  13. Decoding chromatin goes high tech.