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Sample records for phosphoproteome dynamics reveal

  1. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  2. Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

    PubMed Central

    Humphrey, Sean J.; Yang, Guang; Yang, Pengyi; Fazakerley, Daniel J.; Stöckli, Jacqueline; Yang, Jean Y.; James, David E.

    2013-01-01

    Summary A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists. PMID:23684622

  3. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics

    PubMed Central

    Doubleday, Peter F.; Ballif, Bryan A.

    2014-01-01

    Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX) chromatography, coupled to immobilized metal affinity chromatography (IMAC), we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain. PMID:25177544

  4. Phosphoproteomic dynamics of chickpea (Cicer arietinum L.) reveals shared and distinct components of dehydration response.

    PubMed

    Subba, Pratigya; Barua, Pragya; Kumar, Rajiv; Datta, Asis; Soni, Kamlesh Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

    2013-11-01

    Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water, and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (developmentally regulated plasma membrane protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress, further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.

  5. Dynamic Phosphoproteome Analysis of Seedling Leaves in Brachypodium distachyon L. Reveals Central Phosphorylated Proteins Involved in the Drought Stress Response

    PubMed Central

    Yuan, Lin-Lin; Zhang, Ming; Yan, Xing; Bian, Yan-Wei; Zhen, Shou-Min; Yan, Yue-Ming

    2016-01-01

    Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant’s abiotic stress response through phosphorylation modification. PMID:27748408

  6. Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves*

    PubMed Central

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-01-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  7. Phosphoproteome dynamics upon changes in plant water status reveal early events associated with rapid growth adjustment in maize leaves.

    PubMed

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-10-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  8. Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium.

    PubMed

    Soares, Nelson C; Spät, Philipp; Krug, Karsten; Macek, Boris

    2013-06-07

    Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth.

  9. Temporal Dynamics of the Saccharopolyspora erythraea Phosphoproteome*

    PubMed Central

    Licona-Cassani, Cuauhtemoc; Lim, SooA; Marcellin, Esteban; Nielsen, Lars K.

    2014-01-01

    Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest (“metabolic switch”) preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO2 enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of

  10. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation.

    PubMed

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

  11. TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics*

    PubMed Central

    Zhong, Jun; Kim, Min-Sik; Chaerkady, Raghothama; Wu, Xinyan; Huang, Tai-Chung; Getnet, Derese; Mitchell, Christopher J.; Palapetta, Shyam M.; Sharma, Jyoti; O'Meally, Robert N.; Cole, Robert N.; Yoda, Akinori; Moritz, Albrecht; Loriaux, Marc M.; Rush, John; Weinstock, David M.; Tyner, Jeffrey W.; Pandey, Akhilesh

    2012-01-01

    Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets

  12. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

    PubMed Central

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria. PMID:25873915

  13. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

    PubMed Central

    Ficarro, Scott B.; Hutchinson, John N.; Csepanyi-Komi, Roland; Nguyen, Phi T.; Wisniewski, Eva; Sullivan, Jessica; Hofmann, Oliver; Ligeti, Erzsebet; Marto, Jarrod A.; Wagers, Amy J.

    2016-01-01

    Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization. PMID:27365422

  14. Glucose-regulated and drug-perturbed phosphoproteome reveals molecular mechanisms controlling insulin secretion

    PubMed Central

    Sacco, Francesca; Humphrey, Sean J.; Cox, Jürgen; Mischnik, Marcel; Schulte, Anke; Klabunde, Thomas; Schäfer, Matthias; Mann, Matthias

    2016-01-01

    Insulin-secreting beta cells play an essential role in maintaining physiological blood glucose levels, and their dysfunction leads to the development of diabetes. To elucidate the signalling events regulating insulin secretion, we applied a recently developed phosphoproteomics workflow. We quantified the time-resolved phosphoproteome of murine pancreatic cells following their exposure to glucose and in combination with small molecule compounds that promote insulin secretion. The quantitative phosphoproteome of 30,000 sites clustered into three main groups in concordance with the modulation of the three key kinases: PKA, PKC and CK2A. A high-resolution time course revealed key novel regulatory sites, revealing the importance of methyltransferase DNMT3A phosphorylation in the glucose response. Remarkably a significant proportion of these novel regulatory sites is significantly downregulated in diabetic islets. Control of insulin secretion is embedded in an unexpectedly broad and complex range of cellular functions, which are perturbed by drugs in multiple ways. PMID:27841257

  15. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  16. Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties*

    PubMed Central

    Narushima, Yuta; Kozuka-Hata, Hiroko; Koyama-Nasu, Ryo; Tsumoto, Kouhei; Inoue, Jun-ichiro; Akiyama, Tetsu; Oyama, Masaaki

    2016-01-01

    Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation. PMID:26670566

  17. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus.

    PubMed

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y L; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  18. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

    PubMed Central

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y. L.; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription–translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  19. Battle through Signaling between Wheat and the Fungal Pathogen Septoria tritici Revealed by Proteomics and Phosphoproteomics*

    PubMed Central

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

    2013-01-01

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

  20. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  1. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

    PubMed Central

    Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

    2017-01-01

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  2. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  3. Quantitative phosphoproteomics reveals Wee1 kinase as a therapeutic target in a model of proneural glioblastoma

    PubMed Central

    Lescarbeau, Rebecca S.; Lei, Liang; Bakken, Katrina K.; Sims, Peter A.; Sarkaria, Jann N.; Canoll, Peter; White, Forest M.

    2016-01-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an anti-tumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in pre-clinical models of GBM. PMID:27196784

  4. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    PubMed

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

  6. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    PubMed Central

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  7. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network.

    PubMed

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-20

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism.

  8. Brain Membrane Proteome and Phosphoproteome Reveal Molecular Basis Associating with Nursing and Foraging Behaviors of Honeybee Workers.

    PubMed

    Han, Bin; Fang, Yu; Feng, Mao; Hu, Han; Hao, Yue; Ma, Chuan; Huo, Xinmei; Meng, Lifeng; Zhang, Xufeng; Wu, Fan; Li, Jianke

    2017-10-06

    The brain is a vital organ in regulating complex social behaviors of honeybees including learning and memory. Knowledge of how brain membrane proteins and their phosphorylation underlie the age-related behavioral polyethism is still lacking. A hitherto age-resolved brain membrane proteome and phosphoproteome were reported in adult worker bees from two strains of honeybee (Apis mellifera ligustica): Italian bee (ITB) and Royal Jelly bee (RJB), a line selected from ITB for increased RJ outputs over four decades. There were 1079 membrane protein groups identified, and 417 unique phosphosites were located in 179 membrane protein groups mainly phosphorylated by kinase families of MAPKs, CDKs, and CK2. Age-resolved dynamics of brain membrane proteome and phosphoproteome are indicative of their correlation with the neurobiological requirements during the adult life of honeybee workers. To stimulate immature brain cell development in newly emerged bees (NEBs), the enriched functional classes associated with metabolism of carbohydrates, nucleosides, and lipids by the up-regulated proteins suggest their enhanced role in driving cell maturity of the brain. In nurse bees (NBs) and forager bees (FBs), a higher number of membrane proteins and phosphoproteins were expressed as compared with in the young stages, and the enriched signal-transduction-related pathways by the up-regulated proteins suggest their significances in sustaining the intensive information processing during nursing and foraging activities. Notably, RJB has shaped unique membrane proteome and phosphoproteome settings to consolidate nursing and foraging behaviors in response to decades of selection underpinning the elevated RJ yields. In RJB NBs, the enriched pathways of phosphatidylinositol signaling and arachidonic acid metabolism indicate a stronger olfaction sensation in response to larval pheromone stimulation. In RJB FBs, the enriched pathways related to signal processing such as SNARE interactions in

  9. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  10. Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

    PubMed

    Jouy, Florent; Müller, Stephan A; Wagner, Juliane; Otto, Wolfgang; von Bergen, Martin; Tomm, Janina M

    2015-01-01

    Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

  11. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  12. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-05

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype.

  13. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

    PubMed

    Nirujogi, Raja Sekhar; Wright, James D; Manda, Srikanth S; Zhong, Jun; Na, Chan Hyun; Meyerhoff, James; Benton, Bernard; Jabbour, Rabih; Willis, Kristen; Kim, Min-Sik; Pandey, Akhilesh; Sekowski, Jennifer W

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

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

    PubMed Central

    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

  15. Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars*

    PubMed Central

    Pi, Erxu; Qu, Liqun; Hu, Jianwen; Huang, Yingying; Qiu, Lijuan; Lu, Hongfei; Jiang, Bo; Liu, Cong; Peng, Tingting; Zhao, Ying; Wang, Huizhong; Tsai, Sau-Na; Ngai, Saiming; Du, Liqun

    2016-01-01

    Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by

  16. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  17. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

    PubMed

    Bodenmiller, Bernd; Wanka, Stefanie; Kraft, Claudine; Urban, Jörg; Campbell, David; Pedrioli, Patrick G; Gerrits, Bertran; Picotti, Paola; Lam, Henry; Vitek, Olga; Brusniak, Mi-Youn; Roschitzki, Bernd; Zhang, Chao; Shokat, Kevan M; Schlapbach, Ralph; Colman-Lerner, Alejandro; Nolan, Garry P; Nesvizhskii, Alexey I; Peter, Matthias; Loewith, Robbie; von Mering, Christian; Aebersold, Ruedi

    2010-12-21

    The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

  18. Phosphoproteomics Reveals Distinct Modes of Mec1/ATR Signaling During DNA Replication

    PubMed Central

    de Oliveira, Francisco Meirelles Bastos; Kim, Dongsung; Cussiol, Jose Renato; Das, Jishnu; Jeong, Min Cheol; Doerfler, Lillian; Schmidt, Kristina Hildegard; Yu, Haiyuan; Smolka, Marcus Bustamante

    2015-01-01

    SUMMARY The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1’s activation state induced by replication stress. This “replication-correlated” mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor, and is distinguishable from Mec1’s action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress. PMID:25752575

  19. Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance.

    PubMed

    Pi, Zhi; Zhao, Mei-Ling; Peng, Xian-Jun; Shen, Shi-Hua

    2017-04-13

    Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the protein-protein interaction network analysis, a protein kinase and phosphatases hub protein (P1959) were found to be involved in cross-talk between Ca(2+), BR, ABA, and ethylene-mediated signaling pathways. We also highlighted the phosphorylation of BpSIZ1 and BpICE1 possibly impacted on the CBF/DREB-responsive pathway. From these results, we developed a schematic for the chilling tolerance mechanism at phosphorylation level.

  20. Phosphoproteomic analysis reveals the importance of kinase regulation during orbivirus infection.

    PubMed

    Mohl, Bjorn-Patrick; Emmott, Edward; Roy, Polly

    2017-08-29

    Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signalling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analysed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  2. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

    PubMed Central

    Beck, Florian; Geiger, Jörg; Gambaryan, Stepan; Solari, Fiorella A.; Dell’Aica, Margherita; Loroch, Stefan; Mattheij, Nadine J.; Mindukshev, Igor; Pötz, Oliver; Jurk, Kerstin; Burkhart, Julia M.; Fufezan, Christian; Heemskerk, Johan W. M.; Walter, Ulrich

    2017-01-01

    Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein–coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. PMID:28060719

  3. Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

    Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

  4. Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy.

    PubMed

    Chang, Yu-Wang; Chang, Ya-Ting; Wang, Qinchuan; Lin, Jim Jung-Ching; Chen, Yu-Ju; Chen, Chien-Chang

    2013-11-01

    Pressure-overload stress to the heart causes pathological cardiac hypertrophy, which increases the risk of cardiac morbidity and mortality. However, the detailed signaling pathways induced by pressure overload remain unclear. Here we used phosphoproteomics to delineate signaling pathways in the myocardium responding to acute pressure overload and chronic hypertrophy in mice. Myocardial samples at 4 time points (10, 30, 60 min and 2 weeks) after transverse aortic banding (TAB) in mice underwent quantitative phosphoproteomics assay. Temporal phosphoproteomics profiles showed 360 phosphorylation sites with significant regulation after TAB. Multiple mechanical stress sensors were activated after acute pressure overload. Gene ontology analysis revealed differential phosphorylation between hearts with acute pressure overload and chronic hypertrophy. Most interestingly, analysis of the cardiac hypertrophy pathway revealed phosphorylation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) by prohypertrophic kinases. Phosphorylation of DRP1 S622 was confirmed in TAB-treated mouse hearts and phenylephrine (PE)-treated rat neonatal cardiomyocytes. TAB-treated mouse hearts showed phosphorylation-mediated mitochondrial translocation of DRP1. Inhibition of DRP1 with the small-molecule inhibitor mdivi-1 reduced the TAB-induced hypertrophic responses. Mdivi-1 also prevented PE-induced hypertrophic growth and oxygen consumption in rat neonatal cardiomyocytes. We reveal the signaling responses of the heart to pressure stress in vivo and in vitro. DRP1 may be important in the development of cardiac hypertrophy.

  5. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus

    PubMed Central

    Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718

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

    PubMed Central

    Wei, Sijia; Bian, Yangyang; Zhao, Qi; Chen, Sixue; Mao, Jiawei; Song, Chunxia; Cheng, Kai; Xiao, Zhen; Zhang, Chuanfang; Ma, Weimin; Zou, Hanfa; Ye, Mingliang; Dai, Shaojun

    2017-01-01

    Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC) proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway), nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein–protein interaction (PPI) networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain. PMID:28588593

  7. Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

    PubMed

    Chidiac, Rony; Zhang, Ying; Tessier, Sylvain; Faubert, Denis; Delisle, Chantal; Gratton, Jean-Philippe

    2016-05-01

    VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.

  8. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    PubMed Central

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  9. Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

    PubMed Central

    Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O.; Emili, Andrew

    2016-01-01

    Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function. PMID:27742792

  10. Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy.

    PubMed

    Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O; Emili, Andrew

    2016-11-01

    Phospholamban (PLN) plays a central role in Ca(2+) homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2A (SERCA2A) Ca(2+) pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.

  11. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

    Mass Spectrometry (MS)-based phosphoproteomics tools are crucial for understanding the structure and dynamics of signaling networks. Approaches such as affinity purification followed by MS have also been used to elucidate relevant biological questions in health and disease. The study of proteomes and phosphoproteomes as linked systems, rather than research studies of individual proteins, are necessary to understand the functions of phosphorylated and un-phosphorylated proteins under spatial and temporal conditions. Phosphoproteome studies also facilitate drug target protein identification which may be clinically useful in the near future. Here, we provide an overview of general principles of signaling pathways versus phosphorylation. Likewise, we detail chemical phosphoproteomic tools, including pros and cons with examples where these methods have been applied. In addition, basic clues of electrospray ionization and collision induced dissociation fragmentation are detailed in a simple manner for successful phosphoproteomic clinical studies. PMID:23369623

  12. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  13. Phosphoproteomic profiling of the myocyte.

    PubMed

    Edwards, Alistair V G; Cordwell, Stuart J; White, Melanie Y

    2011-10-01

    Protein phosphorylation underpins major cellular processes including energy metabolism, signal transduction, excitation-contraction coupling, apoptosis, and cell survival mechanisms and is thus critical to the myocyte. Targeted approaches, whereby a handful of phosphoproteins are investigated, can suffer from a relatively narrow view of cellular phosphorylation. In contrast, recent technical advances have allowed for the comprehensive documentation of phosphorylation events in complex biological environments, providing a deeper view of the "phosphoproteome." A global, high-throughput characterization of the myocardial phosphoproteome, however, has not yet been achieved. Efficient analysis of phosphorylated proteins and their roles in a dynamic cellular environment requires high-resolution strategies that can identify, localize, and quantify many thousands of phosphorylation sites in a single experiment. Such an approach requires specific enrichment and purification techniques, developed to align with high-end instrumentation for analysis. Cutting-edge phosphoproteomics is no longer restricted to gel-based technology, instead focusing on affinity enrichment prior to liquid chromatography and mass spectrometry. We will describe the best current methods and how they can be applied, as well as the challenges associated with them. We also present current phosphoproteomic investigations in the myocyte and its subcompartments. Although the techniques and instrumentation required to achieve the goal of a myocardial phosphoprotein catalog in physiological and diseased states are highly specialized, the potential biological insight provided by such an approach makes phosphoproteomics an important new avenue of investigation for the cardiovascular researcher.

  14. Proteomic and phosphoproteomic analysis reveals the response and defense mechanism in leaves of diploid wheat T. monococcum under salt stress and recovery.

    PubMed

    Lv, Dong-Wen; Zhu, Geng-Rui; Zhu, Dong; Bian, Yan-Wei; Liang, Xiao-Na; Cheng, Zhi-Wei; Deng, Xiong; Yan, Yue-Ming

    2016-06-30

    Salinity is a major abiotic stress factor affecting crops production and productivity. Triticum monococcum is closely related to Triticum urartu (A(U)A(U)), which is used as a model plant of wheat A genome study. Here, salt stress induced dynamic proteome and phosphoproteome profiling was focused. The T. monococcum seedlings were initially treated with different concentrations of NaCl ranging from 80 to 320mM for 48h followed by a recovery process for 48h prior to proteomic and phosphoproteomic analysis. As a result, a total of 81 spots corresponding to salt stress and recovery were identified by MALDI-TOF/TOF-MS from 2-DE gels. These proteins were mainly involved in regulatory, stress defense, protein folding/assembly/degradation, photosynthesis, carbohydrate metabolism, energy production and transportation, protein metabolism, and cell structure. Pro-Q Diamond staining was used to detect the phosphoproteins. Finally, 20 spots with different phosphorylation levels during salt treatment or recovery compared with controls were identified. A set of potential salt stress response and defense biomarkers was identified, such as cp31BHv, betaine-aldehyde dehydrogenase, leucine aminopeptidase 2, Cu/Zn superoxide dismutase, and 2-Cys peroxiredoxin BAS1, which could lead to a better understanding of the molecular basis of salt response and defense in food crops. Soil salinity reduces the yield of the major crops, which is one of the severest problems in irrigated agriculture worldwide. However, how crops response and defense during different levels of salt treatment and recovery processes is still unclear, especially at the post-translational modification level. T. monococcum is a useful model for common wheat. Thus, proteomic and phosphoproteomic analyses of T. monococcum leaves were performed in our study, which provided novel insights into the underlying salt response and defense mechanisms in wheat and other crops. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Unraveling the phosphoproteome dynamics in mammal mitochondria from a network perspective.

    PubMed

    Padrão, Ana Isabel; Vitorino, Rui; Duarte, José Alberto; Ferreira, Rita; Amado, Francisco

    2013-10-04

    With mitochondrion garnering more attention for its inextricable involvement in pathophysiological conditions, it seems imperative to understand the means by which the molecular pathways harbored in this organelle are regulated. Protein phosphorylation has been considered a central event in cellular signaling and, more recently, in the modulation of mitochondrial activity. Efforts have been made to understand the molecular mechanisms by which protein phosphorylation regulates mitochondrial signaling. With the advances in mass-spectrometry-based proteomics, there is a substantial hope and expectation in the increased knowledge of protein phosphorylation profile and its mode of regulation. On the basis of phosphorylation profiles, attempts have been made to disclose the kinases involved and how they control the molecular processes in mitochondria and, consequently, the cellular outcomes. Still, few studies have focused on mitochondrial phosphoproteome profiling, particularly in diseases. The present study reviews current data on protein phosphorylation profiling in mitochondria, the potential kinases involved and how pathophysiological conditions modulate the mitochondrial phosphoproteome. To integrate data from distinct research papers, we performed network analysis, with bioinformatic tools like Cytoscape, String, and PANTHER taking into consideration variables such as tissue specificity, biological processes, molecular functions, and pathophysiological conditions. For instance, data retrieved from these analyses evidence some homology in the mitochondrial phosphoproteome among liver and heart, with proteins from transport and oxidative phosphorylation clusters particularly susceptible to phosphorylation. A distinct profile was noticed for adipocytes, with proteins form metabolic processes, namely, triglycerides metabolism, as the main targets of phosphorylation. Regarding disease conditions, more phosphorylated proteins were observed in diabetics with some

  16. Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Zhang, Ming; Zhu, Gengrui; Lv, Dongwen; Wang, Yaping; Zhu, Dong; Yan, Yueming

    2017-01-01

    Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. PMID:28194157

  17. Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

    PubMed

    Rinschen, Markus M; Pahmeyer, Caroline; Pisitkun, Trairak; Schnell, Nicole; Wu, Xiongwu; Maaß, Martina; Bartram, Malte P; Lamkemeyer, Tobias; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2015-04-01

    Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion.

    PubMed

    Milani, Renato; Ferreira, Carmen V; Granjeiro, José M; Paredes-Gamero, Edgar J; Silva, Rodrigo A; Justo, Giselle Z; Nader, Helena B; Galembeck, Eduardo; Peppelenbosch, Maikel P; Aoyama, Hiroshi; Zambuzzi, Willian F

    2010-04-01

    Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion-related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3beta during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell-cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems-oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell-substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering.

  19. Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles

    PubMed Central

    Eisenhardt, Anja E.; Sprenger, Adrian; Röring, Michael; Herr, Ricarda; Weinberg, Florian; Köhler, Martin; Braun, Sandra; Orth, Joachim; Diedrich, Britta; Lanner, Ulrike; Tscherwinski, Natalja; Schuster, Simon; Dumaz, Nicolas; Schmidt, Enrico; Baumeister, Ralf; Schlosser, Andreas

    2016-01-01

    B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase. PMID:27034005

  20. Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions.

    PubMed

    Marumoto, Ariane; Milani, Renato; da Silva, Rodrigo A; da Costa Fernandes, Célio Junior; Granjeiro, José Mauro; Ferreira, Carmen V; Peppelenbosch, Maikel P; Zambuzzi, Willian F

    2017-10-01

    The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. Copyright © 2017 Elsevier Inc

  1. Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿ †

    PubMed Central

    Pines, Alex; Kelstrup, Christian D.; Vrouwe, Mischa G.; Puigvert, Jordi C.; Typas, Dimitris; Misovic, Branislav; de Groot, Anton; von Stechow, Louise; van de Water, Bob; Danen, Erik H. J.; Vrieling, Harry; Mullenders, Leon H. F.; Olsen, Jesper V.

    2011-01-01

    Cellular responses to DNA-damaging agents involve the activation of various DNA damage signaling and transduction pathways. Using quantitative and high-resolution tandem mass spectrometry, we determined global changes in protein level and phosphorylation site profiles following treatment of SILAC (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) consensus sequence (S/T-Q motif) was significantly overrepresented among hyperphosphorylated peptides, about half of the >2-fold-upregulated phosphorylation sites based on the consensus sequence were not direct substrates of ATM and ATR. Eleven protein kinases mainly belonging to the mitogen-activated protein kinase (MAPK) family were identified as being regulated in their kinase domain activation loop. The biological importance of three of these kinases (cyclin-dependent kinase 7 [CDK7], Plk1, and KPCD1) in the protection against cisplatin-induced cytotoxicity was demonstrated by small interfering RNA (siRNA)-mediated knockdown. Our results indicate that the cellular response to cisplatin involves a variety of kinases and phosphatases not only acting in the nucleus but also regulating cytoplasmic targets, resulting in extensive cytoskeletal rearrangements. Integration of transcriptomic and proteomic data revealed a poor correlation between changes in the relative levels of transcripts and their corresponding proteins, but a large overlap in affected pathways at the levels of mRNA, protein, and phosphoprotein. This study provides an integrated view of pathways activated by genotoxic stress and deciphers kinases that play a pivotal role in regulating cellular processes other than the DNA damage response. PMID:22006019

  2. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  3. Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs[C][W

    PubMed Central

    van Wijk, Klaas J.; Friso, Giulia; Walther, Dirk; Schulze, Waltraud X.

    2014-01-01

    Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry–based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts. PMID:24894044

  4. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    PubMed Central

    Højlund, Kurt; Bowen, Benjamin P.; Hwang, Hyonson; Flynn, Charles R.; Madireddy, Lohith; Thangiah, Geetha; Langlais, Paul; Meyer, Christian; Mandarino, Lawrence J.; Yi, Zhengping

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. PMID:19764811

  5. Nuclear phosphoproteomics analysis reveals that CDK1/2 are involved in EGF-regulated constitutive pre-mRNA splicing in MDA-MB-468 cells.

    PubMed

    Chen, Xianwei; Guo, Dan; Zhu, Yinghui; Xian, Feng; Liu, Siqi; Wu, Lin; Lou, Xiaomin

    2016-06-01

    The epidermal growth factor (EGF) receptor (EGFR) pathway is one of the most dysregulated and extensively investigated signaling pathways in human cancers and plays important roles in the regulation of nuclear functions through both cytoplasmic and nuclear EGFR pathways. However, the current understanding of the nuclear phosphorylation responses to activated EGFR pathways remains limited. In the present study, phosphoproteomics analysis revealed the increased phosphorylation of 90 nuclear proteins, primarily involved in RNA processing, pre-mRNA splicing and cell cycle regulation, upon EGF stimulation in MDA-MB-468 cells. Cellular splicing assays of the β-globin (HBB) minigene confirmed that EGF induced constitutive pre-mRNA splicing. Further analysis of phosphoproteomics data identified multiple CDK1/2 substrates in pre-mRNA splicing-related proteins, and both CDK1/2 inhibitors and CDK1/2 knockdowns reduced EGF-regulated pre-mRNA splicing. In conclusion, the results of the present study provide evidence that CDK1/2 participate in the regulation of constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells. In this study, we successfully carried out a survey of nuclear phosphorylation changes in response to EGF stimulation. The results from the functional category analysis and pre-mRNA splicing assay strongly indicated that EGFR activation increased constitutive pre-mRNA splicing in MDA-MB-468 cells, revealing additional role of EGFR on regulation of mRNA maturation beyond alternative pre-mRNA splicing reported by previous studies. Furthermore, we found that CDK1/2 participated in constitutive pre-mRNA splicing regulation by EGF in MDA-MB-468 cells. Our study provides new knowledge for understanding the regulation of constitutive pre-mRNA splicing by EGF stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms

    PubMed Central

    Elagamey, Eman; Narula, Kanika; Sinha, Arunima; Aggarwal, Pooja Rani; Ghosh, Sudip; Chakraborty, Niranjan; Chakraborty, Subhra

    2016-01-01

    The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling. PMID:28248230

  7. Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17*

    PubMed Central

    Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. PMID:26499836

  8. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

    PubMed Central

    Yi, Tingfang; Zhai, Bo; Yu, Yonghao; Kiyotsugu, Yoshikawa; Raschle, Thomas; Etzkorn, Manuel; Seo, Hee-Chan; Nagiec, Michal; Luna, Rafael E.; Reinherz, Ellis L.; Blenis, John; Gygi, Steven P.; Wagner, Gerhard

    2014-01-01

    Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4–mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4–mediated phosphoproteome, including construction of kinase–substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy. PMID

  9. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-02-02

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

  10. Phosphoproteomics Reveals Resveratrol-Dependent Inhibition of Akt/mTORC1/S6K1 Signaling

    PubMed Central

    2015-01-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

  11. Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

    PubMed Central

    Kettenbach, Arminja N.; Deng, Lin; Wu, Youjun; Baldissard, Suzanne; Adamo, Mark E.; Gerber, Scott A.; Moseley, James B.

    2015-01-01

    Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets. PMID:25720772

  12. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

    PubMed

    Gioia, Romain; Leroy, Cédric; Drullion, Claire; Lagarde, Valérie; Etienne, Gabriel; Dulucq, Stéphanie; Lippert, Eric; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

  13. Quantitative phosphoproteomics reveals pathways for coordination of cell growth and division by the conserved fission yeast kinase pom1.

    PubMed

    Kettenbach, Arminja N; Deng, Lin; Wu, Youjun; Baldissard, Suzanne; Adamo, Mark E; Gerber, Scott A; Moseley, James B

    2015-05-01

    Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF)

    PubMed Central

    Reiland, Sonja; Finazzi, Giovanni; Endler, Anne; Willig, Adrian; Baerenfaller, Katja; Grossmann, Jonas; Gerrits, Bertran; Rutishauser, Dorothea; Gruissem, Wilhelm; Rochaix, Jean-David; Baginsky, Sacha

    2011-01-01

    Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast. PMID:21768351

  15. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

    PubMed

    Alayev, Anya; Doubleday, Peter F; Berger, Sara Malka; Ballif, Bryan A; Holz, Marina K

    2014-12-05

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.

  16. Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

    PubMed

    Posch, Christian; Sanlorenzo, Martina; Vujic, Igor; Oses-Prieto, Juan A; Cholewa, Brian D; Kim, Sarasa T; Ma, Jeffrey; Lai, Kevin; Zekhtser, Mitchell; Esteve-Puig, Rosaura; Green, Gary; Chand, Shreya; Burlingame, Alma L; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-10-01

    In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

  17. Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation

    PubMed Central

    Rich, Matthew T.; Abbott, Thomas B.; Chung, Lisa; Gulcicek, Erol E.; Stone, Kathryn L.; Colangelo, Christopher M.; Lam, TuKiet T.; Nairn, Angus C.; Taylor, Jane R.

    2016-01-01

    Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance

  18. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  19. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  20. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.

    PubMed

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E

    2015-08-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.

  1. Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation.

    PubMed

    Rich, Matthew T; Abbott, Thomas B; Chung, Lisa; Gulcicek, Erol E; Stone, Kathryn L; Colangelo, Christopher M; Lam, TuKiet T; Nairn, Angus C; Taylor, Jane R; Torregrossa, Mary M

    2016-07-20

    Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for

  2. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells

    PubMed Central

    FANG, YI; ZHANG, QIAN; WANG, XIN; YANG, XUE; WANG, XIANGYU; HUANG, ZHEN; JIAO, YUCHEN; WANG, JING

    2016-01-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  3. Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

    PubMed Central

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-01-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. PMID:25693802

  4. Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

    PubMed Central

    Wang, Yi-Ting; Pan, Szu-Hua; Tsai, Chia-Feng; Kuo, Ting-Chun; Hsu, Yuan-Ling; Yen, Hsin-Yung; Choong, Wai-Kok; Wu, Hsin-Yi; Liao, Yen-Chen; Hong, Tse-Ming; Sung, Ting-Yi; Yang, Pan-Chyr; Chen, Yu-Ju

    2017-01-01

    Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations, most patients develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome and proteome of drug-sensitive and drug-resistant NSCLC cells under gefitinib treatment. The construction of a dose-dependent responsive kinase-substrate network of 1548 phosphoproteins and 3834 proteins revealed CK2-centric modules as the dominant core network for the potential gefitinib resistance-associated proteins. CK2 knockdown decreased cell survival in gefitinib-resistant NSCLCs. Using motif analysis to identify the CK2 core sub-network, we verified that elevated phosphorylation level of a CK2 substrate, HMGA1 was a critical node contributing to EGFR-TKI resistance in NSCLC cell. Both HMGA1 knockdown or mutation of the CK2 phosphorylation site, S102, of HMGA1 reinforced the efficacy of gefitinib in resistant NSCLC cells through reactivation of the downstream signaling of EGFR. Our results delineate the TKI resistance-associated kinase-substrate network, suggesting a potential therapeutic strategy for overcoming TKI-induced resistance in NSCLC. PMID:28290473

  5. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

    Li, Jinna; Silva-Sanchez, Cecilia; Zhang, Tong; Chen, Sixue; Li, Haiying

    2015-01-01

    Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. PMID:26136758

  6. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    PubMed

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-04

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

  7. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    PubMed

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  8. Databases for plant phosphoproteomics.

    PubMed

    Schulze, Waltraud X; Yao, Qiuming; Xu, Dong

    2015-01-01

    Phosphorylation is the most studied posttranslational modification involved in signal transduction in stress responses, development, and growth. In the recent years large-scale phosphoproteomic studies were carried out using various model plants and several growth and stress conditions. Here we present an overview of online resources for plant phosphoproteomic databases: PhosPhAt as a resource for Arabidopsis phosphoproteins, P3DB as a resource expanding to crop plants, and Medicago PhosphoProtein Database as a resource for the model plant Medicago trunculata.

  9. Identifying differentially regulated subnetworks from phosphoproteomic data

    PubMed Central

    2010-01-01

    Background Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. Results Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. Conclusions SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data. PMID:20584295

  10. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

    PubMed Central

    Li, Renfeng; Pinto, Sneha M.; Shaw, Patrick G.; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S.; Pandey, Akhilesh; Hayward, S. Diane

    2015-01-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  11. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation on Keratin-8 in skin squamous cell carcinoma derived cell- line.

    PubMed

    Tiwari, Richa; Sahu, Indrajit; Soni, Bihari Lal; Sathe, Gajanan J; Datta, Keshava K; Thapa, Pankaj; Sinha, Shruti; Vadivel, Chella Krishna; Dhaka, Bharti; Gowda, Harsha; Vaidya, Milind M

    2017-02-07

    Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine(73) (head-domain) and Serine(431) (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed TMT-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1(T14,Y15) , EIF4EBP1(T46,T50) , EIF4B(S422) , AKT1S1T246,S247, CTTN1(T401,S405,) Y421 & CAP1(S307/309) in K8-S73A/D mutant and CTTN1(T401,S405,Y421) , BUB1B(S1043) & CARHSP1(S30,S32) in K8-S431A/D mutants as well as some anonymous phosphosites including MYC(S176) , ZYX(S344) and PNN(S692) could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site- specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies. This article is protected by copyright. All rights reserved.

  12. Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents.

    PubMed

    Ramsubramaniam, Nikhil; Tao, Feng; Li, Shuwei; Marten, Mark R

    2013-12-01

    We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from alpha casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART.

  13. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets

    PubMed Central

    Petsalaki, Evangelia; Helbig, Andreas O.; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P.; Pawson, Tony

    2015-01-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific ‘network wiring’. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib—a tyrosine kinase inhibitor (TKI)—in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  14. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

    Reversible protein phosphorylation serves as a basis for regulating a number of cellular processes. Aberrant activation of kinase signaling pathways is commonly associated with several cancers. Recent developments in phosphoprotein/phosphopeptide enrichment strategies and quantitative mass spectrometry have resulted in robust pipelines for high-throughput characterization of phosphorylation in a global fashion. Today, it is possible to profile site-specific phosphorylation events on thousands of proteins in a single experiment. The potential of this approach is already being realized to characterize signaling pathways that govern oncogenesis. In addition, chemical proteomic strategies have been used to unravel targets of kinase inhibitors, which are otherwise difficult to characterize. This review summarizes various approaches used for analysis of the phosphoproteome in general, and protein kinases in particular, highlighting key cancer phosphoproteomic studies. PMID:20937571

  15. Phosphoproteomics in cereals.

    PubMed

    Yang, Pingfang

    2015-01-01

    Cereals are the most important crop plant supplying staple food throughout the world. The economic importance and continued breeding of crop plants such as rice, maize, wheat, or barley require a detailed scientific understanding of adaptive and developmental processes. Protein phosphorylation is one of the most important regulatory posttranslational modifications and its analysis allows deriving functional and regulatory principles in plants. This minireview summarizes the current knowledge of phosphoproteomic studies in cereals.

  16. Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.

    PubMed

    Franck, William L; Gokce, Emine; Randall, Shan M; Oh, Yeonyee; Eyre, Alex; Muddiman, David C; Dean, Ralph A

    2015-06-05

    The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

  17. Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

    PubMed

    Resjö, Svante; Ali, Ashfaq; Meijer, Harold J G; Seidl, Michael F; Snel, Berend; Sandin, Marianne; Levander, Fredrik; Govers, Francine; Andreasson, Erik

    2014-04-04

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

  18. Phosphoproteome-based kinase activity profiling reveals the critical role of MAP2K2 and PLK1 in neuronal autophagy.

    PubMed

    Chen, Lei-Lei; Wang, Yong-Bo; Song, Ju-Xian; Deng, Wan-Kun; Lu, Jia-Hong; Ma, Li-Li; Yang, Chuan-Bin; Li, Min; Xue, Yu

    2017-09-21

    Recent studies have demonstrated that dysregulation of macroautophagy/autophagy may play a central role in the pathogenesis of neurodegenerative disorders, and the induction of autophagy protects against the toxic insults of aggregate-prone proteins by enhancing their clearance. Thus, autophagy has become a promising therapeutic target against neurodegenerative diseases. In this study, quantitative phosphoproteomic profiling together with a computational analysis was performed to delineate the phosphorylation signaling networks regulated by 2 natural neuroprotective autophagy enhancers, corynoxine (Cory) and corynoxine B (Cory B). To identify key regulators, namely, protein kinases, we developed a novel network-based algorithm of in silico Kinome Activity Profiling (iKAP) to computationally infer potentially important protein kinases from phosphorylation networks. Using this algorithm, we observed that Cory or Cory B potentially regulated several kinases. We predicted and validated that Cory, but not Cory B, downregulated a well-documented autophagy kinase, RPS6KB1/p70S6K (ribosomal protein S6 kinase, polypeptide 1). We also discovered 2 kinases, MAP2K2/MEK2 (mitogen-activated protein kinase kinase 2) and PLK1 (polo-like kinase 1), to be potentially upregulated by Cory, whereas the siRNA-mediated knockdown of Map2k2 and Plk1 significantly inhibited Cory-induced autophagy. Furthermore, Cory promoted the clearance of Alzheimer disease-associated APP (amyloid β [A4] precursor protein) and Parkinson disease-associated SNCA/α-synuclein (synuclein, α) by enhancing autophagy, and these effects were dramatically diminished by the inhibition of the kinase activities of MAP2K2 and PLK1. As a whole, our study not only developed a powerful method for the identification of important regulators from the phosphoproteomic data but also identified the important role of MAP2K2 and PLK1 in neuronal autophagy.

  19. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

    2013-01-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  20. Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms.

    PubMed

    Stulemeijer, Iris J E; Joosten, Matthieu H A J; Jensen, Ole N

    2009-03-01

    An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Phosphopeptides were quantified using a label-free approach based on the MS peak areas. We identified 12 phosphopeptides for which the abundance changed upon HR initiation, as compared to control seedlings. Our results suggest that photosynthetic activity is specifically suppressed in a phosphorylation-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling. We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR.

  1. SILAC-based temporal phosphoproteomics.

    PubMed

    Francavilla, Chiara; Hekmat, Omid; Blagoev, Blagoy; Olsen, Jesper V

    2014-01-01

    In recent years, thanks to advances in Mass Spectrometry (MS)-based quantitative proteomics, studies on signaling pathways have moved from a detailed description of individual components to system-wide analysis of entire signaling cascades, also providing spatio-temporal views of intracellular pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Analysis of enriched peptides on Orbitrap-based MS results in comprehensive and accurate reconstruction of temporal changes of signaling networks.

  2. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

    SciTech Connect

    Chan, Chi Yuet X’avia; Gritsenko, Marina A.; Smith, Richard D.; Qian, Wei-Jun

    2016-03-17

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been revealed in various human diseases. Kinases and their cognate inhibitors have been hotspot for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for the disease of interest. In this review, we will highlight recent advances in phosphoproteomics, the current state-of-the-art of the technologies, and the challenges and future perspectives of this research area. Finally, we will underscore some exemplary applications of phosphoproteomics in diabetes research.

  3. Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

    PubMed Central

    Shin, Ryoung; Alvarez, Sophie; Burch, Adrien Y.; Jez, Joseph M.; Schachtman, Daniel P.

    2007-01-01

    SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes. PMID:17404219

  4. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

    PubMed Central

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  5. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light

  6. Automatic generation of predictive dynamic models reveals nuclear phosphorylation as the key Msn2 control mechanism.

    PubMed

    Sunnåker, Mikael; Zamora-Sillero, Elias; Dechant, Reinhard; Ludwig, Christina; Busetto, Alberto Giovanni; Wagner, Andreas; Stelling, Joerg

    2013-05-28

    Predictive dynamical models are critical for the analysis of complex biological systems. However, methods to systematically develop and discriminate among systems biology models are still lacking. We describe a computational method that incorporates all hypothetical mechanisms about the architecture of a biological system into a single model and automatically generates a set of simpler models compatible with observational data. As a proof of principle, we analyzed the dynamic control of the transcription factor Msn2 in Saccharomyces cerevisiae, specifically the short-term mechanisms mediating the cells' recovery after release from starvation stress. Our method determined that 12 of 192 possible models were compatible with available Msn2 localization data. Iterations between model predictions and rationally designed phosphoproteomics and imaging experiments identified a single-circuit topology with a relative probability of 99% among the 192 models. Model analysis revealed that the coupling of dynamic phenomena in Msn2 phosphorylation and transport could lead to efficient stress response signaling by establishing a rate-of-change sensor. Similar principles could apply to mammalian stress response pathways. Systematic construction of dynamic models may yield detailed insight into nonobvious molecular mechanisms.

  7. Insights Regarding Fungal Phosphoproteomic Analysis.

    PubMed

    Ribeiro, Liliane F C; Chelius, Cynthia L; Harris, Steven D; Marten, Mark R

    2017-03-10

    Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.

  8. Eye Movements Reveal Dynamics of Task Control

    ERIC Educational Resources Information Center

    Mayr, Ulrich; Kuhns, David; Rieter, Miranda

    2013-01-01

    With the goal to determine the cognitive architecture that underlies flexible changes of control settings, we assessed within-trial and across-trial dynamics of attentional selection by tracking of eye movements in the context of a cued task-switching paradigm. Within-trial dynamics revealed a switch-induced, discrete delay in onset of…

  9. Eye Movements Reveal Dynamics of Task Control

    ERIC Educational Resources Information Center

    Mayr, Ulrich; Kuhns, David; Rieter, Miranda

    2013-01-01

    With the goal to determine the cognitive architecture that underlies flexible changes of control settings, we assessed within-trial and across-trial dynamics of attentional selection by tracking of eye movements in the context of a cued task-switching paradigm. Within-trial dynamics revealed a switch-induced, discrete delay in onset of…

  10. Methods for and Insights from Phosphoproteome Analysis in Marine Microbes

    NASA Astrophysics Data System (ADS)

    Held, N. A.; Saito, M. A.; McIlvin, M.

    2016-02-01

    Phosphorylation, the dynamic addition of a phosphate group to specific amino acids, is a key regulator of protein activity in both prokaryotes and eukaryotes. Protein phosphorylation is known to modulate nutrient acquisition, metabolism, growth and reproduction in model organisms, yet little is known about the role of phosphorylation marine organisms. Recent developments in LC-MS/MS make it possible to identify phosphorylation events in the proteome. We tested various methods in marine bacteria and developed a simple approach to phosphoproteome analysis. We then applied this method to cultured isolates of Prochlorococcus and diatom-associated Alteromonas sp. BB2AT2. We began by comparing the phosphoproteomes of these organisms in exponential and stationary phase growth. We conducted iterative experiments to assess completeness of our analysis, similar to the rarefaction approach used to determine sequence depth in ecology. We also explored semi-quantitative changes in protein phosphorylation when cells were subject to phosphate deplete media and/or phosphatase inhibitors. These early studies demonstrate the promise of phosphoproteomics to advance our understanding of bacterial biochemistry and microbe-environment interactions.

  11. SILAC for global phosphoproteomic analysis.

    PubMed

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

    Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes.

  12. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  13. Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

    PubMed

    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.

  14. SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

    PubMed Central

    Zhang, H; Xu, Y; Filipovic, A; Lit, L C; Koo, C-Y; Stebbing, J; Giamas, G

    2013-01-01

    Background: We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. Methods: A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. Results: Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. Conclusion: Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity. PMID:24129246

  15. Functional Phosphoproteomic Analysis Reveals That a Serine-62-Phosphorylated Isoform of Ethylene Response Factor110 Is Involved in Arabidopsis Bolting1[C][W][OA

    PubMed Central

    Zhu, Lin; Liu, Dandan; Li, Yaojun; Li, Ning

    2013-01-01

    Ethylene is a major plant hormone that plays an important role in regulating bolting, although the underlying molecular mechanism is not well understood. In this study, we report the novel finding that the serine-62 (Ser-62) phosphorylation of Ethylene Response Factor110 (ERF110) is involved in the regulation of bolting time. The gene expression and posttranslational modification (phosphorylation) of ERF110 were analyzed among ethylene-response mutants and ERF110 RNA-interfering knockout lines of Arabidopsis (Arabidopsis thaliana). Physiological and biochemical studies revealed that the Ser-62 phosphorylation of ERF110 was closely related to bolting time, that is, the ethylene-enhanced gene expression of ERF110 and the decreased Ser-62 phosphorylation of the ERF110 protein in Arabidopsis. The expression of a flowering homeotic APETALA1 gene was up-regulated by the Ser-62-phosphorylated isoform of the ERF110 transcription factor, which was necessary but not sufficient for normal bolting. The gene expression and phosphorylation of ERF110 were regulated by ethylene via both Ethylene-Insensitive2-dependent and -independent pathways, which constitute a dual-and-opposing mechanism of action for ethylene in the regulation of Arabidopsis bolting. PMID:23188807

  16. Phosphoproteome Discovery in Human Biological Fluids

    PubMed Central

    Giorgianni, Francesco; Beranova-Giorgianni, Sarka

    2016-01-01

    Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented. PMID:28248247

  17. Phosphoproteome Discovery in Human Biological Fluids.

    PubMed

    Giorgianni, Francesco; Beranova-Giorgianni, Sarka

    2016-12-01

    Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented.

  18. An Initial Characterization of the Serum Phosphoproteome

    PubMed Central

    Zhou, Weidong; Ross, Mark M.; Tessitore, Alessandra; Ornstein, David; VanMeter, Amy; Liotta, Lance A.; Petricoin, Emanuel F.

    2009-01-01

    Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. Using this approach we identified ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate. PMID:19824718

  19. Multiplexed Detection of O-GlcNAcome, Phosphoproteome, and Whole Proteome within the Same Gel

    PubMed Central

    Cieniewski-Bernard, Caroline; Dupont, Erwan; Deracinois, Barbara; Lambert, Matthias; Bastide, Bruno

    2014-01-01

    The cellular diversity of proteins results in part from their post-translational modifications. Among all of them, the O-GlcNAcylation is an atypical glycosylation, more similar to phosphorylation than classical glycosylations. Highly dynamic, reversible, and exclusively localized on cytosolic, nuclear, and mitochondrial proteins, O-GlcNAcylation is known to regulate almost all if not all cellular processes. Fundamental for the cell life, O-GlcNAcylation abnormalities are involved in the etiology of several inherited diseases. Assessing to O-GlcNAcylation pattern will permit to get relevant data about the role of O-GlcNAcylation in cell physiology. To get understanding about the role of O-GlcNAcylation, as also considering its interplay with phosphorylation, the O-GlcNAc profiling remains a real challenge for the community of proteomists/glycoproteomists. The development of multiplexed proteomics based on fluorescent detection of proteins permits to go further in the understanding of the proteome complexity. We propose herein a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. In particular, we investigated the phosphoproteome through the ProQ Diamond staining, while the whole proteome was visualized through Sypro Ruby staining, or after the labeling of proteins with a T-Dye fluorophore. The O-GlcNAcome was revealed by the way of the Click chemistry and the azide–alkyne cycloaddition of a fluorophore on GlcNAc moieties. This method permits, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome. PMID:25389416

  20. Phosphoproteomic Analysis of Liver Homogenates

    PubMed Central

    Demirkan, Gokhan; Salomon, Arthur R.; Gruppuso, Philip A.

    2013-01-01

    Summary Regulation of protein function via reversible phosphorylation is an essential component of cell signaling. Our ability to understand complex phosphorylation networks in the physiological context of a whole organism or tissue remains limited. This is largely due to the technical challenge of isolating serine/threonine phosphorylated peptides from a tissue sample. In the present study, we developed a phosphoproteomic strategy to purify and identify phosphopeptides from a tissue sample by employing protein gel filtration, protein SAX (strong anion exchange) and SCX (strong cation exchange) chromatography, peptide SCX chromatography and TiO2 affinity purification. By applying this strategy to the mass spectrometry-based analysis of rat liver homogenates, we were able to identify with high confidence and quantify over four thousand unique phosphopeptides. Finally, the reproducibility of our methodology was demonstrated by its application to analysis of the mammalian Target of Rapamycin (mTOR) signaling pathways in liver samples obtained from rats in which hepatic mTOR was activated by refeeding following a period of fasting. PMID:22903715

  1. Neutron Imaging Reveals Internal Plant Hydraulic Dynamics

    SciTech Connect

    Warren, Jeffrey; Bilheux, Hassina Z; Kang, Misun; Voisin, Sophie; Cheng, Chu-Lin; Horita, Jusuke; Perfect, Edmund

    2013-01-01

    Many terrestrial ecosystem processes are constrained by water availability and transport within the soil. Knowledge of plant water fluxes is thus critical for assessing mechanistic processes linked to biogeochemical cycles, yet resolution of root structure and xylem water transport dynamics has been a particularly daunting task for the ecologist. Through neutron imaging, we demonstrate the ability to non-invasively monitor individual root functionality and water fluxes within Zea mays L. (maize) and Panicum virgatum L. (switchgrass) seedlings growing in a sandy medium. Root structure and growth were readily imaged by neutron radiography and neutron computed tomography. Seedlings were irrigated with water or deuterium oxide and imaged through time as a growth lamp was cycled on to alter leaf demand for water. Sub-millimeter scale resolution reveals timing and magnitudes of root water uptake, redistribution within the roots, and root-shoot hydraulic linkages, relationships not well characterized by other techniques.

  2. Sawfishes stealth revealed using computational fluid dynamics.

    PubMed

    Bradney, D R; Davidson, A; Evans, S P; Wueringer, B E; Morgan, D L; Clausen, P D

    2017-02-27

    Detailed computational fluid dynamics simulations for the rostrum of three species of sawfish (Pristidae) revealed that negligible turbulent flow is generated from all rostra during lateral swipe prey manipulation and swimming. These results suggest that sawfishes are effective stealth hunters that may not be detected by their teleost prey's lateral line sensory system during pursuits. Moreover, during lateral swipes, the rostra were found to induce little velocity into the surrounding fluid. Consistent with previous data of sawfish feeding behaviour, these data indicate that the rostrum is therefore unlikely to be used to stir up the bottom to uncover benthic prey. Whilst swimming with the rostrum inclined at a small angle to the horizontal, the coefficient of drag of the rostrum is relatively low and the coefficient of lift is zero.

  3. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  4. Phosphoproteomics and molecular cardiology: techniques, applications and challenges.

    PubMed

    Sun, Zeyu; Hamilton, Karyn L; Reardon, Kenneth F

    2012-09-01

    Protein phosphorylation has been widely documented as a key regulatory and signaling mechanism associated with many cardiac diseases. Recent advances in phosphoproteomic technologies such as phosphopeptide enrichment, novel mass spectrometry applications, and bioinformatic tools have resulted in high-throughput identification and quantitation of protein phosphorylation in a global manner. This review summarizes mainstream phosphoproteomic workflows and highlights the most recent applications of phosphoproteomics used in a range of molecular cardiology research.

  5. Neutron Imaging Reveals Internal Plant Hydraulic Dynamics

    NASA Astrophysics Data System (ADS)

    Warren, J.; Bilheux, H.; Kang, M.; Voisin, S.; Cheng, C.; Horita, J.; Perfect, E.

    2011-12-01

    In situ quantification of soil-plant water fluxes have not been fully successful due to a lack of non-destructive techniques capable of revealing roots or water fluxes at relevant spatial scales. Neutron imaging is a unique non-invasive tool that can assess sub-millimeter scale material properties and transport in situ, and which has been successfully applied to characterize soil and plant water status. Here, we have applied neutron radiography and tomography to quantify water transport through individual maize roots in response to internal plant demand. Zea mays seedlings were grown for 10 days in Flint silica sand within 2.6 cm diameter Al chambers. Using a reactor-based neutron source at Oak Ridge National Laboratory (HFIR), water fluxes were tracked through the maize soil-root systems by collecting consecutive neutron radiographs over a 12 h period following irrigation with D2O. D has a much lower neutron attenuation than H, thus D2O displacement of existing H2O within the plant vascular system, or influx of D2O into previously dry tissue or soil is readily tracked by changes in image intensity through time. Plant water release and uptake was regulated by periodically cycling on a high-intensity grow light. From each maize replicate, selected regions of interest (ROI) were delineated around individual roots, root free soil, stem and leaf segments. Changes in ROI were tracked through time to reveal patterns of water flux. The hydration of root and stem tissue cycled in response to illumination; root water content often increased during darkness, then decreased with illumination as water was transported from the root into the stem. Relative root-shoot hydration through time illustrates the balance between demand, storage capacity and uptake, which varies depending on root characteristics and its localized soil environment. The dynamic transport of water between soil, individual roots, stems and leaves was readily visualized and quantified illustrating the value

  6. Planetary Interior Structure Revealed by Spin Dynamics

    NASA Astrophysics Data System (ADS)

    Margot, J.; Peale, S. J.; Jurgens, R. F.; Slade, M. A.; Holin, I. V.

    2002-12-01

    The spin state of a planet depends on the distribution of mass within the interior, gradual and discrete changes in its moments of inertia, dissipation mechanisms at the surface and below, and external torques. Detailed measurements of the spin dynamics can therefore reveal much about planetary interior structure, interactions at the core-mantle and atmosphere-surface boundaries, and mass redistribution events. Studies of the spin precession, polar wobble, and length of day variations have been used to determine Earth's moments of inertia and rigidity and to study the effects of atmospheric angular momentum changes, post-glacial rebound, and large earthquakes. In planetary investigations the spin measurements are particularly important because other means of constraining interior properties require in-situ or orbiting sensors (e.g. seismometers, magnetometers, and Doppler tracking of spacecraft). Here we describe the successful implementation of a new Earth-based radar technique (Holin, 1992) that provides spin state measurements with unprecedented accuracy. Our first observations were designed to characterize Mercury's core. Peale (1976) showed that the measurement of four quantities (the obliquity of the planet, the amplitude of its longitude librations, and the second-degree gravitational harmonics) are sufficient to determine the size and state of Mercury's core. The existence of a molten core would place strong constraints on the thermal and rotational histories of the planet, with profound implications for the composition and rotation state of the planet at the time of formation. A solid core would have a fundamental impact on theories of planetary magnetic field generation. We observed Mercury with the Goldstone radar and the Green Bank Telescope in May-June 2002. We illuminated the planet with a monochromatic signal, recorded the scattered power at the two antennas, and cross-correlated the echoes in the time domain. We obtained strong correlations which

  7. Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

    PubMed

    Verma, Renu; Pinto, Sneha Maria; Patil, Arun Hanumana; Advani, Jayshree; Subba, Pratigya; Kumar, Manish; Sharma, Jyoti; Dey, Gourav; Ravikumar, Raju; Buggi, Shashidhar; Satishchandra, Parthasarathy; Sharma, Kusum; Suar, Mrutyunjay; Tripathy, Srikanth Prasad; Chauhan, Devendra Singh; Gowda, Harsha; Pandey, Akhilesh; Gandotra, Sheetal; Prasad, Thottethodi Subrahmanya Keshava

    2017-03-20

    Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

  8. Dispersion of Response Times Reveals Cognitive Dynamics

    PubMed Central

    Holden, John G.; Van Orden, Guy C.; Turvey, Michael T.

    2013-01-01

    Trial to trial variation in word pronunciation times exhibits 1/f scaling. One explanation is that human performances are consequent on multiplicative interactions among interdependent processes – interaction dominant dynamics. This article describes simulated distributions of pronunciation times in a further test for multiplicative interactions and interdependence. Individual participant distributions of ≈1100 word pronunciation times are successfully mimicked for each participant in combinations of lognormal and power law behavior. Successful hazard function simulations generalize these results to establish interaction dominant dynamics, in contrast with component dominant dynamics, as a likely mechanism for cognitive activity. PMID:19348544

  9. Revealing networks from dynamics: an introduction

    NASA Astrophysics Data System (ADS)

    Timme, Marc; Casadiego, Jose

    2014-08-01

    What can we learn from the collective dynamics of a complex network about its interaction topology? Taking the perspective from nonlinear dynamics, we briefly review recent progress on how to infer structural connectivity (direct interactions) from accessing the dynamics of the units. Potential applications range from interaction networks in physics, to chemical and metabolic reactions, protein and gene regulatory networks as well as neural circuits in biology and electric power grids or wireless sensor networks in engineering. Moreover, we briefly mention some standard ways of inferring effective or functional connectivity.

  10. Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis.

    PubMed

    Polat, Ayse Nur; Karayel, Özge; Giese, Sven H; Harmanda, Büşra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y; Özlü, Nurhan

    2015-09-04

    Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

  11. Deuterium reveals the dynamics of notch activation.

    PubMed

    Raphael, Kopan

    2011-04-13

    Notch activation requires unfolding of a juxtamembrane negative regulatory domain (NRR). Tiyanont et al. (2011) analyzed the dynamics of NRR unfolding in the presence of EGTA. As predicted from the crystal structure and deletion analyses, the lin-Notch repeats unfold first, facilitating access by ADAM proteases. Surprisingly, the heterodimerization domain remains stable.

  12. Using Dynamic Graphs to Reveal Student Reasoning

    ERIC Educational Resources Information Center

    Lassak, Marshall

    2009-01-01

    Using dynamic graphs, future secondary mathematics teachers were able to represent and communicate their understanding of a brief mathematical investigation in a way that a symbolic proof of the problem could not. Four different student work samples are discussed. (Contains 6 figures.)

  13. Dispersion of Response Times Reveals Cognitive Dynamics

    ERIC Educational Resources Information Center

    Holden, John G.; Van Orden, Guy C.; Turvey, Michael T.

    2009-01-01

    Trial-to-trial variation in word-pronunciation times exhibits 1/f scaling. One explanation is that human performances are consequent on multiplicative interactions among interdependent processes-interaction dominant dynamics. This article describes simulated distributions of pronunciation times in a further test for multiplicative interactions and…

  14. Integrated quantitative analysis of the phosphoproteome and transcriptome in tamoxifen-resistant breast cancer.

    PubMed

    Oyama, Masaaki; Nagashima, Takeshi; Suzuki, Takashi; Kozuka-Hata, Hiroko; Yumoto, Noriko; Shiraishi, Yuichi; Ikeda, Kazuhiro; Kuroki, Yoko; Gotoh, Noriko; Ishida, Takanori; Inoue, Satoshi; Kitano, Hiroaki; Okada-Hatakeyama, Mariko

    2011-01-07

    Quantitative phosphoproteome and transcriptome analysis of ligand-stimulated MCF-7 human breast cancer cells was performed to understand the mechanisms of tamoxifen resistance at a system level. Phosphoproteome data revealed that WT cells were more enriched with phospho-proteins than tamoxifen-resistant cells after stimulation with ligands. Surprisingly, decreased phosphorylation after ligand perturbation was more common than increased phosphorylation. In particular, 17β-estradiol induced down-regulation in WT cells at a very high rate. 17β-Estradiol and the ErbB ligand heregulin induced almost equal numbers of up-regulated phospho-proteins in WT cells. Pathway and motif activity analyses using transcriptome data additionally suggested that deregulated activation of GSK3β (glycogen-synthase kinase 3β) and MAPK1/3 signaling might be associated with altered activation of cAMP-responsive element-binding protein and AP-1 transcription factors in tamoxifen-resistant cells, and this hypothesis was validated by reporter assays. An examination of clinical samples revealed that inhibitory phosphorylation of GSK3β at serine 9 was significantly lower in tamoxifen-treated breast cancer patients that eventually had relapses, implying that activation of GSK3β may be associated with the tamoxifen-resistant phenotype. Thus, the combined phosphoproteome and transcriptome data set analyses revealed distinct signal transcription programs in tumor cells and provided a novel molecular target to understand tamoxifen resistance.

  15. Peptide crystal simulations reveal hidden dynamics

    PubMed Central

    Janowski, Pawel A.; Cerutti, David S.; Holton, James; Case, David A.

    2013-01-01

    Molecular dynamics simulations of biomolecular crystals at atomic resolution have the potential to recover information on dynamics and heterogeneity hidden in the X-ray diffraction data. We present here 9.6 microseconds of dynamics in a small helical peptide crystal with 36 independent copies of the unit cell. The average simulation structure agrees with experiment to within 0.28 Å backbone and 0.42 Å all-atom rmsd; a model refined against the average simulation density agrees with the experimental structure to within 0.20 Å backbone and 0.33 Å all-atom rmsd. The R-factor between the experimental structure factors and those derived from this unrestrained simulation is 23% to 1.0 Å resolution. The B-factors for most heavy atoms agree well with experiment (Pearson correlation of 0.90), but B-factors obtained by refinement against the average simulation density underestimate the coordinate fluctuations in the underlying simulation where the simulation samples alternate conformations. A dynamic flow of water molecules through channels within the crystal lattice is observed, yet the average water density is in remarkable agreement with experiment. A minor population of unit cells is characterized by reduced water content, 310 helical propensity and a gauche(−) side-chain rotamer for one of the valine residues. Careful examination of the experimental data suggests that transitions of the helices are a simulation artifact, although there is indeed evidence for alternate valine conformers and variable water content. This study highlights the potential for crystal simulations to detect dynamics and heterogeneity in experimental diffraction data, as well as to validate computational chemistry methods. PMID:23631449

  16. Phosphoproteomics by Mass Spectrometry: insights, implications, applications, and limitations

    PubMed Central

    Mayya, Viveka; Han, David K.

    2010-01-01

    Summary Phosphorylation of proteins is a predominant reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective we discuss four aspects of phosphoproteomics; namely insights and implications from recently published phosphoproteomic studies, and applications and limitations of current phosphoproteomic strategies. As about 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis based investigations. Finally we discuss strategies to measure stoichiometry of phosphorylation in a proteome-wide manner which is not provided by current phosphoproteomic approaches. PMID:19929607

  17. Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network.

    PubMed

    Kumar, Rajiv; Kumar, Amit; Subba, Pratigya; Gayali, Saurabh; Barua, Pragya; Chakraborty, Subhra; Chakraborty, Niranjan

    2014-06-13

    Nucleus, the control centre of eukaryotic cell, houses most of the genetic machineries required for gene expression and their regulation. Post translational modifications of proteins, particularly phosphorylation control a wide variety of cellular processes but its functional connectivity, in plants, is still elusive. This study profiled the nuclear phosphoproteome of a grain legume, chickpea, to gain better understanding of such event. Intact nuclei were isolated from 3-week-old seedlings using two independent methods, and nuclear proteins were resolved by 2-DE. In a separate set of experiments, phosphoproteins were enriched using IMAC method and resolved by 1-DE. The separated proteins were stained with phosphospecific Pro-Q Diamond stain. Proteomic analyses led to the identification of 107 putative phosphoproteins, of which 86 were non-redundant. Multiple sites of phosphorylation were predicted on several key elements, which included both regulatory and functional proteins. The analysis revealed an array of phosphoproteins, presumably involved in a variety of cellular functions, viz., protein folding (24%), signalling and gene regulation (22%), DNA replication, repair and modification (16%), and metabolism (13%), among others. These results represent the first nucleus-specific phosphoproteome map of a non-model legume, which would provide insights into the possible function of protein phosphorylation in plants. Chickpea is grown over 10 million hectares of land worldwide, and global production hovers around 8.5 million metric tons annually. Despite its nutritional merits, it is often referred to as 'orphan' legume and has remained outside the realm of large-scale functional genomics studies. While current chickpea genome initiative has primarily focused on sequence information and functional annotation, proteomics analyses are limited. It is thus important to study the proteome of the cell organelle particularly the nucleus, which harbors most of the genetic

  18. AVHRR imagery reveals Antarctic ice dynamics

    NASA Technical Reports Server (NTRS)

    Bindschadler, Robert A.; Vornberger, Patricia L.

    1990-01-01

    A portion of AVHRR data taken on December 5, 1987 at 06:15 GMT over a part of Antarctica is used here to show that many of the most significant dynamic features of ice sheets can be identified by a careful examination of AVHRR imagery. The relatively low resolution of this instrument makes it ideal for obtaining a broad view of the ice sheets, while its wide swath allows coverage of areas beyond the reach of high-resolution imagers either currently in orbit or planned. An interpretation is given of the present data, which cover the area of ice streams that drain the interior of the West Antarctic ice sheet into the Ross Ice Shelf.

  19. AVHRR imagery reveals Antarctic ice dynamics

    SciTech Connect

    Bindschadler, R.A.; Vornberger, P.L. STX Corp., Lanham, MD )

    1990-06-01

    A portion of AVHRR data taken on December 5, 1987 at 06:15 GMT over a part of Antarctica is used here to show that many of the most significant dynamic features of ice sheets can be identified by a careful examination of AVHRR imagery. The relatively low resolution of this instrument makes it ideal for obtaining a broad view of the ice sheets, while its wide swath allows coverage of areas beyond the reach of high-resolution imagers either currently in orbit or planned. An interpretation is given of the present data, which cover the area of ice streams that drain the interior of the West Antarctic ice sheet into the Ross Ice Shelf. 21 refs.

  20. Human dynamics revealed through Web analytics.

    PubMed

    Gonçalves, Bruno; Ramasco, José J

    2008-08-01

    The increasing ubiquity of Internet access and the frequency with which people interact with it raise the possibility of using the Web to better observe, understand, and monitor several aspects of human social behavior. Web sites with large numbers of frequently returning users are ideal for this task. If these sites belong to companies or universities, their usage patterns can furnish information about the working habits of entire populations. In this work, we analyze the properly anonymized logs detailing the access history to Emory University's Web site. Emory is a medium-sized university located in Atlanta, Georgia. We find interesting structure in the activity patterns of the domain and study in a systematic way the main forces behind the dynamics of the traffic. In particular, we find that linear preferential linking, priority-based queuing, and the decay of interest for the contents of the pages are the essential ingredients to understand the way users navigate the Web.

  1. Human dynamics revealed through Web analytics

    NASA Astrophysics Data System (ADS)

    Gonçalves, Bruno; Ramasco, José J.

    2008-08-01

    The increasing ubiquity of Internet access and the frequency with which people interact with it raise the possibility of using the Web to better observe, understand, and monitor several aspects of human social behavior. Web sites with large numbers of frequently returning users are ideal for this task. If these sites belong to companies or universities, their usage patterns can furnish information about the working habits of entire populations. In this work, we analyze the properly anonymized logs detailing the access history to Emory University’s Web site. Emory is a medium-sized university located in Atlanta, Georgia. We find interesting structure in the activity patterns of the domain and study in a systematic way the main forces behind the dynamics of the traffic. In particular, we find that linear preferential linking, priority-based queuing, and the decay of interest for the contents of the pages are the essential ingredients to understand the way users navigate the Web.

  2. Wigner flow reveals topological order in quantum phase space dynamics.

    PubMed

    Steuernagel, Ole; Kakofengitis, Dimitris; Ritter, Georg

    2013-01-18

    The behavior of classical mechanical systems is characterized by their phase portraits, the collections of their trajectories. Heisenberg's uncertainty principle precludes the existence of sharply defined trajectories, which is why traditionally only the time evolution of wave functions is studied in quantum dynamics. These studies are quite insensitive to the underlying structure of quantum phase space dynamics. We identify the flow that is the quantum analog of classical particle flow along phase portrait lines. It reveals hidden features of quantum dynamics and extra complexity. Being constrained by conserved flow winding numbers, it also reveals fundamental topological order in quantum dynamics that has so far gone unnoticed.

  3. Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.

    PubMed

    Beckers, Gerold J M; Hoehenwarter, Wolfgang; Röhrig, Horst; Conrath, Uwe; Weckwerth, Wolfram

    2014-01-01

    In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it allows unbiased localization, and site-specific quantification, of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affinity chromatography (MOAC) represents a robust and highly selective approach for the identification and site-specific quantification of low abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides. This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.

  4. Phosphoproteomic analysis of protein phosphorylation networks in Tetrahymena thermophila, a model single-celled organism.

    PubMed

    Tian, Miao; Chen, Xiulan; Xiong, Qian; Xiong, Jie; Xiao, Chuanle; Ge, Feng; Yang, Fuquan; Miao, Wei

    2014-02-01

    Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

  5. Revealing physical interaction networks from statistics of collective dynamics.

    PubMed

    Nitzan, Mor; Casadiego, Jose; Timme, Marc

    2017-02-01

    Revealing physical interactions in complex systems from observed collective dynamics constitutes a fundamental inverse problem in science. Current reconstruction methods require access to a system's model or dynamical data at a level of detail often not available. We exploit changes in invariant measures, in particular distributions of sampled states of the system in response to driving signals, and use compressed sensing to reveal physical interaction networks. Dynamical observations following driving suffice to infer physical connectivity even if they are temporally disordered, are acquired at large sampling intervals, and stem from different experiments. Testing various nonlinear dynamic processes emerging on artificial and real network topologies indicates high reconstruction quality for existence as well as type of interactions. These results advance our ability to reveal physical interaction networks in complex synthetic and natural systems.

  6. Revealing physical interaction networks from statistics of collective dynamics

    PubMed Central

    Nitzan, Mor; Casadiego, Jose; Timme, Marc

    2017-01-01

    Revealing physical interactions in complex systems from observed collective dynamics constitutes a fundamental inverse problem in science. Current reconstruction methods require access to a system’s model or dynamical data at a level of detail often not available. We exploit changes in invariant measures, in particular distributions of sampled states of the system in response to driving signals, and use compressed sensing to reveal physical interaction networks. Dynamical observations following driving suffice to infer physical connectivity even if they are temporally disordered, are acquired at large sampling intervals, and stem from different experiments. Testing various nonlinear dynamic processes emerging on artificial and real network topologies indicates high reconstruction quality for existence as well as type of interactions. These results advance our ability to reveal physical interaction networks in complex synthetic and natural systems. PMID:28246630

  7. Increasing phosphoproteomic coverage through sequential digestion by complementary proteases

    PubMed Central

    Gilmore, Jason M.; Kettenbach, Arminja N.; Gerber, Scott A.

    2011-01-01

    Protein phosphorylation is a reversible post-translational modification known to regulate protein function, subcellular localization, complex formation, and protein degradation. Detailed phosphoproteomic information is critical to kinomic studies of signal transduction and for elucidation of cancer biomarkers, such as in non-small cell lung adenocarcinoma, where phosphorylation is commonly dysregulated. However, the collection and analysis of phosphorylation data remains a difficult problem. The low concentrations of phosphopeptides in complex biological mixtures as well as challenges inherent in their chemical nature have limited phosphoproteomic characterization and some phosphorylation sites are inaccessible by traditional workflows. We developed a sequential digestion method using complementary proteases, Glu-C and trypsin, to increase phosphoproteomic coverage and supplement traditional approaches. The sequential digestion method is more productive than workflows utilizing only Glu-C and we evaluated the orthogonality of the sequential digestion method relative to replicate trypsin-based analyses. Finally, we demonstrate the ability of the sequential digestion method to access new regions of the phosphoproteome by comparison to existing public phosphoproteomic databases. Our approach increases coverage of the human lung cancer phosphoproteome by accessing both new phosphoproteins and novel phosphorylation site information. PMID:22002561

  8. Phosphoproteomic Analysis of Isolated Mitochondria in Yeast.

    PubMed

    Renvoisé, Margaux; Bonhomme, Ludovic; Davanture, Marlène; Zivy, Michel; Lemaire, Claire

    2017-01-01

    Mitochondria play a central role in cellular energy metabolism and cell death. Deregulation of mitochondrial functions is associated with several human pathologies (neurodegenerative diseases, neuromuscular diseases, type II diabetes, obesity, cancer). The steadily increasing number of identified mitochondrial phosphoproteins, kinases, and phosphatases in recent years suggests that reversible protein phosphorylation plays an important part in the control of mitochondrial processes. In addition, many mitochondrial phosphoproteins probably still remain to be identified, considering that 30% of proteins are expected to be phosphorylated in eukaryotes. In this chapter, we describe two procedures for the analysis of the mitochondrial phosphoproteome. The first one is a qualitative method that combines blue native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-BN/SDS-PAGE) and specific phosphoprotein staining. The second one is a quantitative approach that associates mitochondrial peptide labeling, phosphopeptide enrichment, and mass spectrometry.

  9. Revealing the Effects of Cognitive Education Programmes through Dynamic Assessment

    ERIC Educational Resources Information Center

    Tzuriel, David

    2011-01-01

    The major objective of this paper is to demonstrate the effectiveness of dynamic assessment (DA) in revealing outcomes of cognitive education programmes. Three programmes based on "mediated learning experience" theory are reviewed: "Feuerstein's Instrumental Enrichment", "Bright Start", and "Peer Mediation with…

  10. Lighting Up T Lymphocyte Signaling with Quantitative Phosphoproteomics.

    PubMed

    Álvarez-Salamero, Candelas; Castillo-González, Raquel; Navarro, María N

    2017-01-01

    Phosphorylation is the most abundant post-translational modification, regulating several aspects of protein and cell function. Quantitative phosphoproteomics approaches have expanded the scope of phosphorylation analysis enabling the quantification of changes in thousands of phosphorylation sites simultaneously in two or more conditions. These approaches offer a global view of the impact of cellular perturbations such as extracellular stimuli or gene ablation in intracellular signaling networks. Such great potential also brings on a new challenge: to identify, among the thousands of phosphorylations found in global phosphoproteomics studies, the small subset of site-specific phosphorylations expected to be functionally relevant. This review focus on updating and integrating findings on T lymphocyte signaling generated using global phosphoproteomics approaches, drawing attention on the biological relevance of the obtained data.

  11. Topological structure dynamics revealing collective evolution in active nematics.

    PubMed

    Shi, Xia-qing; Ma, Yu-qiang

    2013-01-01

    Topological defects frequently emerge in active matter like bacterial colonies, cytoskeleton extracts on substrates, self-propelled granular or colloidal layers and so on, but their dynamical properties and the relations to large-scale organization and fluctuations in these active systems are seldom touched. Here we reveal, through a simple model for active nematics using self-driven hard elliptic rods, that the excitation, annihilation and transportation of topological defects differ markedly from those in non-active media. These dynamical processes exhibit strong irreversibility in active nematics in the absence of detailed balance. Moreover, topological defects are the key factors in organizing large-scale dynamic structures and collective flows, resulting in multi-spatial temporal effects. These findings allow us to control the self-organization of active matter through topological structures.

  12. Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

    PubMed

    Moreira, Douglas de Souza; Pescher, Pascale; Laurent, Christine; Lenormand, Pascal; Späth, Gerald F; Murta, Silvane M F

    2015-09-01

    Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. IKAP: A heuristic framework for inference of kinase activities from Phosphoproteomics data.

    PubMed

    Mischnik, Marcel; Sacco, Francesca; Cox, Jürgen; Schneider, Hans-Christoph; Schäfer, Matthias; Hendlich, Manfred; Crowther, Daniel; Mann, Matthias; Klabunde, Thomas

    2016-02-01

    Phosphoproteomics measurements are widely applied in cellular biology to detect changes in signalling dynamics. However, due to the inherent complexity of phosphorylation patterns and the lack of knowledge on how phosphorylations are related to functions, it is often not possible to directly deduce protein activities from those measurements. Here, we present a heuristic machine learning algorithm that infers the activities of kinases from Phosphoproteomics data using kinase-target information from the PhosphoSitePlus database. By comparing the estimated kinase activity profiles to the measured phosphosite profiles, it is furthermore possible to derive the kinases that are most likely to phosphorylate the respective phosphosite. We apply our approach to published datasets of the human cell cycle generated from HeLaS3 cells, and insulin signalling dynamics in mouse hepatocytes. In the first case, we estimate the activities of 118 at six cell cycle stages and derive 94 new kinase-phosphosite links that can be validated through either database or motif information. In the second case, the activities of 143 kinases at eight time points are estimated and 49 new kinase-target links are derived. The algorithm is implemented in Matlab and be downloaded from github. It makes use of the Optimization and Statistics toolboxes. https://github.com/marcel-mischnik/IKAP.git. marcel.mischnik@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae.

    PubMed

    Renvoisé, Margaux; Bonhomme, Ludovic; Davanture, Marlène; Valot, Benoit; Zivy, Michel; Lemaire, Claire

    2014-06-25

    The yeast Saccharomyces cerevisiae is a facultative aerobe able to adapt its metabolism according to the carbon substrate. The mechanisms of these adaptations involve at least partly the mitochondria but are not yet well understood. To address the possible role of protein phosphorylation event in their regulation, it is necessary in a first instance to determine precisely the phosphorylation sites that show changes depending on the carbon source. In this aim we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media. Label free quantitative analysis of protein accumulation revealed significant variation of 176 mitochondrial proteins including 108 proteins less accumulated in glucose medium than in lactate and galactose media. We also showed that the responses to galactose and glucose are not similar. Stable isotope dimethyl labeling allowed the quantitative comparison of phosphorylation levels between the different growth conditions. This study enlarges significantly the map of yeast mitochondrial phosphosites as 670 phosphorylation sites were identified, of which 214 were new and quantified. Above all, we showed that 90 phosphosites displayed a significant variation according to the medium and that variation of phosphorylation level is site-dependent. This proteomic and phosphoproteomic study is the first extensive study providing quantitative data on phosphosites responses to different carbon substrates independent of the variations of protein quantities in the yeast S. cerevisiae mitochondria. The significant changes observed in the level of phosphorylation according to the carbon substrate open the way to the study of the regulation of mitochondrial proteins by phosphorylation in fermentative and respiratory media. In addition, the identification of a large number of new phosphorylation sites show that the characterization of

  15. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

    SciTech Connect

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese RW; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolic, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-11-11

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e., roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag 8-plex ITRAQ, enriched using Ni-NTA magnetic beads and subjected to nRPLC-MS/MS analysis using HCD and decision tree guided CID/ETD strategy. A total of 1,625 unique phosphopeptides, spanning 1,659 non-redundant phosphorylation sites, were detected from 1,126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5 fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  16. Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib.

    PubMed

    Vyse, Simon; McCarthy, Frank; Broncel, Malgorzata; Paul, Angela; Wong, Jocelyn P; Bhamra, Amandeep; Huang, Paul H

    2017-08-24

    Acquired drug resistance impacts the majority of patients being treated with tyrosine kinase inhibitors (TKIs) and remains a key challenge in modern anti-cancer therapy. The lack of clinically effective therapies to overcome resistance represents an unmet need. Understanding the signalling that drives drug resistance will facilitate the development of new salvage therapies to treat patients with secondary TKI resistance. In this study, we utilise mass spectrometry to characterise the global phosphoproteomic alterations that accompany the acquisition of resistance to two FDA-approved TKIs, pazopanib and dasatinib, in the A204 rhabdoid tumour cell line. Our analysis finds that only 6% and 9.7% of the quantified phosphoproteome is altered upon the acquisition of pazopanib and dasatinib resistance, respectively. Pazopanib resistant cells display elevated phosphorylation in cytoskeletal regulatory pathways while dasatinib resistant cells show an upregulation of the insulin receptor/IGF-1R signalling pathway. Drug response profiling rediscovers several previously reported vulnerabilities associated with pazopanib and dasatinib resistance and identifies a new dependency to the second generation HSP90 inhibitor NVP-AUY-922. This study provides a useful resource detailing the candidate signalling determinants of acquired TKI resistance; and reveals a therapeutic approach of inhibiting HSP90 function as a means of salvage therapy to overcome pazopanib and dasatinib resistance. Pazopanib and dasatinib are tyrosine kinase inhibitors (TKIs) approved for the treatment of multiple cancer types. Patients who are treated with these drugs are prone to the development of drug resistance and consequently tumour relapse. Here we use quantitative phosphoproteomics to characterise the signalling pathways which are enriched in cells that have acquired resistance to these two drugs. Furthermore, targeted drug screens were used to identify salvage therapies capable of overcoming pazopanib

  17. Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum.

    PubMed

    Nguyen, Tran Hong Nha; Brechenmacher, Laurent; Aldrich, Joshua T; Clauss, Therese R; Gritsenko, Marina A; Hixson, Kim K; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolić, Ljiljana; Xu, Dong; Nguyen, Henry T; Stacey, Gary

    2012-11-01

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  18. Versatile nanocomposites in phosphoproteomics: a review.

    PubMed

    Najam-ul-Haq, Muhammad; Jabeen, Fahmida; Hussain, Dilshad; Saeed, Adeela; Musharraf, Syed Ghulam; Huck, Christian W; Bonn, Günther K

    2012-10-17

    Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.

  19. Shotguns in the front line: phosphoproteomics in plants.

    PubMed

    Nakagami, Hirofumi; Sugiyama, Naoyuki; Ishihama, Yasushi; Shirasu, Ken

    2012-01-01

    The emergence of 'shotgun proteomics' has paved the way for high-throughput proteome analysis, by which thousands of proteins can be identified simultaneously from complex samples. Although the shotgun approach has the potential to monitor many different post-translational modifications, further technological development is needed to enrich each post-translational 'modificome'. Large-scale in vivo phosphorylation site mapping, so-called shotgun phosphoproteomics, has become feasible in various organisms, including plants, owing to recent technological breakthroughs. Shotgun phosphoproteomics is not a mature technology, but progress has been rapid. In this review, we highlight the scope and limitations of current methods, and some key technological issues in this field.

  20. NMR reveals a dynamic allosteric pathway in thrombin

    PubMed Central

    Handley, Lindsey D.; Fuglestad, Brian; Stearns, Kyle; Tonelli, Marco; Fenwick, R. Bryn; Markwick, Phineus R. L.; Komives, Elizabeth A.

    2017-01-01

    Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R1, R2, 15N-{1H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with μs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na+-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain μs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form. PMID:28059082

  1. Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

    PubMed Central

    Horst, Walter Johannes

    2013-01-01

    Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

  2. Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.).

    PubMed

    Yang, Zhong-Bao; Eticha, Dejene; Führs, Hendrik; Heintz, Dimitri; Ayoub, Daniel; Van Dorsselaer, Alain; Schlingmann, Barbara; Rao, Idupulapati Madhusudana; Braun, Hans-Peter; Horst, Walter Johannes

    2013-12-01

    Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure.

  3. Revealing the properties of plant defensins through dynamics.

    PubMed

    Valente, Ana Paula; de Paula, Viviane Silva; Almeida, Fabio C L

    2013-09-13

    Defensins are potent, ancient natural antibiotics that are present in organisms ranging from lower organisms to humans. Although the structures of several defensins have been well characterized, the dynamics of only a few have been studied. This review discusses the diverse dynamics of two plant defensins for which the structure and dynamics have been characterized, both in the free state and in the presence of target membranes. Multiple motions are observed in loops and in secondary structure elements and may be related to twisting or breathing of the α-helix and β-sheet. This complex behavior is altered in the presence of an interface and is responsive to the presence of the putative target. The stages of membrane recognition and disruption can be mapped over a large time scale range, demonstrating that defensins in solution exist as an ensemble of different conformations, a subset of which is selected upon membrane binding. Therefore, studies on the dynamics have revealed that defensins interact with membranes through a mechanism of conformational selection.

  4. High-frequency microrheology reveals cytoskeleton dynamics in living cells

    NASA Astrophysics Data System (ADS)

    Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

    2017-08-01

    Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

  5. Ultrafast cooling reveals microsecond-scale biomolecular dynamics.

    PubMed

    Polinkovsky, Mark E; Gambin, Yann; Banerjee, Priya R; Erickstad, Michael J; Groisman, Alex; Deniz, Ashok A

    2014-12-17

    The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10-15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations.

  6. Phosphoproteomic Analysis of Protein Phosphorylation Networks in the Hypopharyngeal Gland of Honeybee Workers (Apis mellifera ligustica).

    PubMed

    Qi, Yuping; Fan, Pei; Hao, Yue; Han, Bin; Fang, Yu; Feng, Mao; Cui, Ziyou; Li, Jianke

    2015-11-06

    The hypopharyngeal gland (HG) in honeybee workers changes functions according to physiological age in the bee colony from producing royal jelly (RJ) in nurse bees to digestive enzymes in foragers. The same set of secretory cells expresses different genes or proteins to create these age-dependent changes; however, it is unknown precisely how the phosphorylation network regulates physiological differences across the development of the adult worker HG. We employed high-accuracy mass-spectrometry-based proteomics to survey phosphoproteome changes in the newly emerged, nurse, and forager bees. Overall, 941, 1322, and 1196 phosphorylation sites matching 1007, 1353, and 1199 phosphopeptides from 549, 720, and 698 phosphoproteins were identified in the three ages of the HG, respectively. Specialized, interconnected phosphorylation networks within each age were found by comparing protein abundance and phosphorylation levels. This illustrates that many proteins are regulated by phosphorylation independent of their expression levels. Furthermore, proteins in key biological processes and pathways were dynamically phosphorylated with age development, including the centrosome cycle, mitotic spindle elongation, macromolecular complex disassembly, and ribosome, indicating that phosphorylation tunes protein activity to optimize cellular behavior of the HG over time. Moreover, complementary protein and phosphoprotein expression is required to support the unique physiology of secretory activity in the HG. This reported data set of the honeybee phosphoproteome significantly improves our understanding of a range of regulatory mechanisms controlling a variety of cellular processes and will serve as a valuable resource for those studying the honeybee and other insects.

  7. Quantitative phosphoproteomic analysis of early seed development in rice (Oryza sativa L.).

    PubMed

    Qiu, Jiehua; Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Lin, Haiyan; Liu, Qing; Zhang, Wen; Li, Zhiyong; Nallamilli, Babi R; Zhang, Jian

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development.

  8. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

    Protein phosphorylation is a complex regulatory event that is involved in the signaling networks that affect virtually every cellular process. The protein phosphorylation may be a novel source for discovering biomarkers and drug targets. However, a systematic analysis of the phosphoproteome in patients with SLE has not been performed. To clarify the pathogenesis of systemic lupus erythematosus (SLE), we compared phosphoprotein expression in PBMCs from SLE patients and normal subjects using proteomics analyses. Phosphopeptides were enriched using TiO2 from PBMCs isolated from 15 SLE patients and 15 healthy subjects and then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. A total of 1035 phosphorylation sites corresponding to 618 NCBI-annotated genes were identified in SLE patients compared with normal subjects. Differentially expressed proteins, peptides and phosphorylation sites were then subjected to bioinformatics analyses. Gene ontology(GO) and pathway analyses showed that nucleic acid metabolism, cellular component organization, transport and multicellular organismal development pathways made up the largest proportions of the differentially expressed genes. Pathway analyses showed that the mitogen-activated protein kinase (MAPK) signaling pathway and actin cytoskeleton regulators made up the largest proportions of the metabolic pathways. Network analysis showed that rous sarcoma oncogene (SRC), v-rel reticuloendotheliosis viral oncogene homolog A (RELA), histone deacetylase (HDA1C) and protein kinase C, delta (PRKCD) play important roles in the stability of the network. These data suggest that aberrant protein phosphorylation may contribute to SLE pathogenesis. PMID:23285258

  9. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    PubMed

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  10. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  11. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    PubMed Central

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  12. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

    PubMed

    Rhoads, Timothy W; Prasad, Aman; Kwiecien, Nicholas W; Merrill, Anna E; Zawack, Kelson; Westphall, Michael S; Schroeder, Frank C; Kimble, Judith; Coon, Joshua J

    2015-11-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

  13. HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

    PubMed

    Shevchuk, Olga; Abidi, Nada; Klawonn, Frank; Wissing, Josef; Nimtz, Manfred; Kugler, Christian; Steinert, Michael; Goldmann, Torsten; Jänsch, Lothar

    2014-11-07

    Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap-frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins, and investigating their biochemical properties, covered subcellular localizations, and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap-frozen tissues. Thus, HOPE-treated tissues match clinical demands in both histology and retrospective proteome analyses of patient samples by proteomics.

  14. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana

    PubMed Central

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  15. Memory in motion: movement dynamics reveal memory strength.

    PubMed

    Papesh, Megan H; Goldinger, Stephen D

    2012-10-01

    Recognition memory is typically examined as a discrete end-state, describable by static variables, such as accuracy, response time, and confidence. In the present study, we combined real-time mouse-tracking with subsequent, overt confidence estimates to examine the dynamic nature of memory decisions. By examining participants' streaming x-, y- mouse coordinates during recognition decisions, we observed that movement trajectories revealed underlying response confidence. More confident decisions were associated with shorter decision times and more linear response trajectories. Less confident decisions were made slowly, with increased trajectory curvature. Statistical indices of curvature and decision times, including area-under-the-curve and time to maximum deviation, suggested that memory strength relates to response dynamics. Whether participants were correct or incorrect, old responses showed a stronger correspondence between mouse trajectories and confidence, relative to new responses. We suggest that people subjectively experience a correspondence between feelings of memory and feelings of confidence; that subjective experience reveals itself in real-time decision processes, as suggested by sequential sampling models of recognition decisions.

  16. Ananke: temporal clustering reveals ecological dynamics of microbial communities

    PubMed Central

    Rohwer, Robin R.; Perrie, Jonathan; McMahon, Katherine D.

    2017-01-01

    Taxonomic markers such as the 16S ribosomal RNA gene are widely used in microbial community analysis. A common first step in marker-gene analysis is grouping genes into clusters to reduce data sets to a more manageable size and potentially mitigate the effects of sequencing error. Instead of clustering based on sequence identity, marker-gene data sets collected over time can be clustered based on temporal correlation to reveal ecologically meaningful associations. We present Ananke, a free and open-source algorithm and software package that complements existing sequence-identity-based clustering approaches by clustering marker-gene data based on time-series profiles and provides interactive visualization of clusters, including highlighting of internal OTU inconsistencies. Ananke is able to cluster distinct temporal patterns from simulations of multiple ecological patterns, such as periodic seasonal dynamics and organism appearances/disappearances. We apply our algorithm to two longitudinal marker gene data sets: faecal communities from the human gut of an individual sampled over one year, and communities from a freshwater lake sampled over eleven years. Within the gut, the segregation of the bacterial community around a food-poisoning event was immediately clear. In the freshwater lake, we found that high sequence identity between marker genes does not guarantee similar temporal dynamics, and Ananke time-series clusters revealed patterns obscured by clustering based on sequence identity or taxonomy. Ananke is free and open-source software available at https://github.com/beiko-lab/ananke. PMID:28966891

  17. Coupled nucleotide covariations reveal dynamic RNA interaction patterns.

    PubMed Central

    Gultyaev, A P; Franch, T; Gerdes, K

    2000-01-01

    Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such "coupled" covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts. PMID:11105748

  18. Stochastic heart-rate model can reveal pathologic cardiac dynamics

    NASA Astrophysics Data System (ADS)

    Kuusela, Tom

    2004-03-01

    A simple one-dimensional Langevin-type stochastic difference equation can simulate the heart-rate fluctuations in a time scale from minutes to hours. The model consists of a deterministic nonlinear part and a stochastic part typical of Gaussian noise, and both parts can be directly determined from measured heart-rate data. Data from healthy subjects typically exhibit the deterministic part with two or more stable fixed points. Studies of 15 congestive heart-failure subjects reveal that the deterministic part of pathologic heart dynamics has no clear stable fixed points. Direct simulations of the stochastic model for normal and pathologic cases can produce statistical parameters similar to those of real subjects. Results directly indicate that pathologic situations simplify the heart-rate control system.

  19. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  20. Rapid Phosphoproteomic and Transcriptomic Changes in the Rhizobia-legume Symbiosis*

    PubMed Central

    Rose, Christopher M.; Venkateshwaran, Muthusubramanian; Volkening, Jeremy D.; Grimsrud, Paul A.; Maeda, Junko; Bailey, Derek J.; Park, Kwanghyun; Howes-Podoll, Maegen; den Os, Désirée; Yeun, Li Huey; Westphall, Michael S.; Sussman, Michael R.; Ané, Jean-Michel; Coon, Joshua J.

    2012-01-01

    Symbiotic associations between legumes and rhizobia usually commence with the perception of bacterial lipochitooligosaccharides, known as Nod factors (NF), which triggers rapid cellular and molecular responses in host plants. We report here deep untargeted tandem mass spectrometry-based measurements of rapid NF-induced changes in the phosphorylation status of 13,506 phosphosites in 7739 proteins from the model legume Medicago truncatula. To place these phosphorylation changes within a biological context, quantitative phosphoproteomic and RNA measurements in wild-type plants were compared with those observed in mutants, one defective in NF perception (nfp) and one defective in downstream signal transduction events (dmi3). Our study quantified the early phosphorylation and transcription dynamics that are specifically associated with NF-signaling, confirmed a dmi3-mediated feedback loop in the pathway, and suggested “cryptic” NF-signaling pathways, some of them being also involved in the response to symbiotic arbuscular mycorrhizal fungi. PMID:22683509

  1. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  2. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

    PubMed

    Fortuin, Suereta; Tomazella, Gisele G; Nagaraj, Nagarjuna; Sampson, Samantha L; Gey van Pittius, Nicolaas C; Soares, Nelson C; Wiker, Harald G; de Souza, Gustavo A; Warren, Robin M

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

  3. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

    PubMed

    Locard-Paulet, Marie; Lim, Lindsay; Veluscek, Giulia; McMahon, Kelly; Sinclair, John; van Weverwijk, Antoinette; Worboys, Jonathan D; Yuan, Yinyin; Isacke, Clare M; Jørgensen, Claus

    2016-02-09

    The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

  4. Quantitative phosphoproteomic analysis using iTRAQ method.

    PubMed

    Asano, Tomoya; Nishiuchi, Takumi

    2014-01-01

    The MAPK (mitogen-activated kinase) cascade plays important roles in plant perception of and reaction to developmental and environmental cues. Phosphoproteomics are useful to identify target proteins regulated by MAPK-dependent signaling pathway. Here, we introduce the quantitative phosphoproteomic analysis using a chemical labeling method. The isobaric tag for relative and absolute quantitation (iTRAQ) method is a MS-based technique to quantify protein expression among up to eight different samples in one experiment. In this technique, peptides were labeled by some stable isotope-coded covalent tags. We perform quantitative phosphoproteomics comparing Arabidopsis wild type and a stress-responsive mapkk mutant after phytotoxin treatment. To comprehensively identify the downstream phosphoproteins of MAPKK, total proteins were extracted from phytotoxin-treated wild-type and mapkk mutant plants. The phosphoproteins were purified by Pro-Q(®) Diamond Phosphoprotein Enrichment Kit and were digested with trypsin. Resulting peptides were labeled with iTRAQ reagents and were quantified and identified by MALDI TOF/TOF analyzer. We identified many phosphoproteins that were decreased in the mapkk mutant compared with wild type.

  5. Temporal dynamics of reward processing revealed by magnetoencephalography.

    PubMed

    Doñamayor, Nuria; Marco-Pallarés, Josep; Heldmann, Marcus; Schoenfeld, M Ariel; Münte, Thomas F

    2011-12-01

    Monetary gains and losses in gambling situations are associated with a distinct electroencephalographic signature: in the event-related potentials (ERPs), a mediofrontal feedback-related negativity (FRN) is seen for losses, whereas oscillatory activity shows a burst of in the θ-range for losses and in the β-range for gains. We used whole-head magnetoencephalography to pinpoint the magnetic counterparts of these effects in young healthy adults and explore their evolution over time. On each trial, participants bet on one of two visually presented numbers (25 or 5) by button-press. Both numbers changed color: if the chosen number turned green (red), it indicated a gain (loss) of the corresponding sum in Euro cent. For losses, we found the magnetic correlate of the FRN extending between 230 and 465 ms. Source localization with low-resolution electromagnetic tomography indicated a first generator in posterior cingulate cortex with subsequent activity in the anterior cingulate cortex. Importantly, this effect was sensitive to the magnitude of the monetary loss (25 cent > 5 cent). Later activation was also found in the right insula. Time-frequency analysis revealed a number of oscillatory components in the theta, alpha, and high-beta/low-gamma bands associated to gains, and in the high-beta band, associated to the magnitude of the loss. All together, these effects provide a more fine-grained picture of the temporal dynamics of the processing of monetary rewards and losses in the brain.

  6. Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach

    SciTech Connect

    Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C.

    2014-03-22

    Mo-dependent nitrogenase catalyzes the biological reduction of N2 to 2NH3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel not previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.

  7. Cytoplasmic dynamics reveals two modes of nucleoid-dependent mobility.

    PubMed

    Stylianidou, Stella; Kuwada, Nathan J; Wiggins, Paul A

    2014-12-02

    It has been proposed that forces resulting from the physical exclusion of macromolecules from the bacterial nucleoid play a central role in organizing the bacterial cell, yet this proposal has not been quantitatively tested. To investigate this hypothesis, we mapped the generic motion of large protein complexes in the bacterial cytoplasm through quantitative analysis of thousands of complete cell-cycle trajectories of fluorescently tagged ectopic MS2-mRNA complexes. We find the motion of these complexes in the cytoplasm is strongly dependent on their spatial position along the long axis of the cell, and that their dynamics are consistent with a quantitative model that requires only nucleoid exclusion and membrane confinement. This analysis also reveals that the nucleoid increases the mobility of MS2-mRNA complexes, resulting in a fourfold increase in diffusion coefficients between regions of the lowest and highest nucleoid density. These data provide strong quantitative support for two modes of nucleoid action: the widely accepted mechanism of nucleoid exclusion in organizing the cell and a newly proposed mode, in which the nucleoid facilitates rapid motion throughout the cytoplasm.

  8. Phosphoproteomic Analysis Identifies Signaling Pathways Regulated by Curcumin in Human Colon Cancer Cells.

    PubMed

    Sato, Tatsuhiro; Higuchi, Yutaka; Shibagaki, Yoshio; Hattori, Seisuke

    2017-09-01

    Curcumin, a major polyphenol of the spice turmeric, acts as a potent chemopreventive and chemotherapeutic agent in several cancer types, including colon cancer. Although various proteins have been shown to be affected by curcumin, how curcumin exerts its anticancer activity is not fully understood. Phosphoproteomic analyses were performed using SW480 and SW620 human colon cancer cells to identify curcumin-affected signaling pathways. Curcumin inhibited the growth of the two cell lines in a dose-dependent manner. Thirty-nine curcumin-regulated phosphoproteins were identified, five of which are involved in cancer signaling pathways. Detailed analyses revealed that the mTORC1 and p53 signaling pathways are main targets of curcumin. Our results provide insight into the molecular mechanisms of the anticancer activities of curcumin and future molecular targets for its clinical application. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  9. Deciphering the acute cellular phosphoproteome response to irradiation with X-rays, protons and carbon ions.

    PubMed

    Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

    2017-03-16

    Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Since radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of

  10. Phosphoproteomic Analysis of the Highly-Metastatic Hepatocellular Carcinoma Cell Line, MHCC97-H

    PubMed Central

    Tian, Miaomiao; Cheng, Han; Wang, Zhiqiang; Su, Na; Liu, Zexian; Sun, Changqing; Zhen, Bei; Hong, Xuechuan; Xue, Yu; Xu, Ping

    2015-01-01

    Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing. PMID:25690035

  11. Machine learning of global phosphoproteomic profiles enables discrimination of direct versus indirect kinase substrates.

    PubMed

    Kanshin, Evgeny; Giguere, Sebastien; Cheng, Jing; Tyers, Michael D; Thibault, Pierre

    2017-03-06

    Mass spectrometry allows quantification of tens of thousands of phosphorylation sites from minute amounts of cellular material. Despite this wealth of information, our understanding of phosphorylation-based signaling is limited, in part because it is not possible to deconvolute substrate phosphorylation that is directly mediated by a particular kinase versus phosphorylation that is mediated by downstream kinases. Here, we describe a framework for assignment of direct in-vivo kinase substrates using a combination of selective chemical inhibition, quantitative phosphoproteomics, and machine learning techniques. Our workflow allows classification of phosphorylation events following inhibition of an analog-sensitive kinase into kinase-independent effects of the inhibitor, direct effects on cognate substrates and indirect effects mediated by downstream kinases or phosphatases. We applied this method to identify many direct targets of Cdc28 and Snf1 kinases in the budding yeast S. cerevisiae. Global phosphoproteome analysis of acute time-series demonstrated that dephosphorylation of direct kinase substrates occurs more rapidly compared to indirect substrates, both after inhibitor treatment and under a physiological nutrient shift in wild-type cells. Mutagenesis experiments revealed a high proportion of functionally relevant phosphorylation sites on Snf1 targets. For example, Snf1 itself was inhibited through autophosphorylation on S391 and new phosphosites were discovered that modulate the activity of the Reg1 regulatory subunit of the Glc7 phosphatase and the Gal83 β-subunit of SNF1 complex. This methodology applies to any kinase for which a functional analog sensitive version can be constructed to facilitate the dissection of the global phosphorylation network.

  12. Dynamics of the Wulong landslide revealed by broadband seismic records

    NASA Astrophysics Data System (ADS)

    Li, Zhengyuan; Huang, Xinghui; Xu, Qiang; Yu, Dan; Fan, Junyi; Qiao, Xuejun

    2017-02-01

    The catastrophic Wulong landslide occurred at 14:51 (Beijing time, UTC+8) on 5 June 2009, in Wulong Prefecture, Southwest China. This rockslide occurred in a complex topographic environment. Seismic signals generated by this event were recorded by the seismic network deployed in the surrounding area, and long-period signals were extracted from 8 broadband seismic stations within 250 km to obtain source time functions by inversion. The location of this event was simultaneously acquired using a stepwise refined grid search approach, with an error of 2.2 km. The estimated source time functions reveal that, according to the movement parameters, this landslide could be divided into three stages with different movement directions, velocities, and increasing inertial forces. The sliding mass moved northward, northeastward and northward in the three stages, with average velocities of 6.5, 20.3, and 13.8 m/s, respectively. The maximum movement velocity of the mass reached 35 m/s before the end of the second stage. The basal friction coefficients were relatively small in the first stage and gradually increasing; large in the second stage, accompanied by the largest variability; and oscillating and gradually decreasing to a stable value, in the third stage. Analysis shows that the movement characteristics of these three stages are consistent with the topography of the sliding zone, corresponding to the northward initiation, eastward sliding after being stopped by the west wall, and northward debris flowing after collision with the east slope of the Tiejianggou valley. The maximum movement velocity of the sliding mass results from the largest height difference of the west slope of the Tiejianggou valley. The basal friction coefficients of the three stages represent the thin weak layer in the source zone, the dramatically varying topography of the west slope of the Tiejianggou valley, and characteristics of the debris flow along the Tiejianggou valley. Based on the above

  13. Laser altimetry reveals complex pattern of Greenland Ice Sheet dynamics.

    PubMed

    Csatho, Beata M; Schenk, Anton F; van der Veen, Cornelis J; Babonis, Gregory; Duncan, Kyle; Rezvanbehbahani, Soroush; van den Broeke, Michiel R; Simonsen, Sebastian B; Nagarajan, Sudhagar; van Angelen, Jan H

    2014-12-30

    We present a new record of ice thickness change, reconstructed at nearly 100,000 sites on the Greenland Ice Sheet (GrIS) from laser altimetry measurements spanning the period 1993-2012, partitioned into changes due to surface mass balance (SMB) and ice dynamics. We estimate a mean annual GrIS mass loss of 243 ± 18 Gt ⋅ y(-1), equivalent to 0.68 mm ⋅ y(-1) sea level rise (SLR) for 2003-2009. Dynamic thinning contributed 48%, with the largest rates occurring in 2004-2006, followed by a gradual decrease balanced by accelerating SMB loss. The spatial pattern of dynamic mass loss changed over this time as dynamic thinning rapidly decreased in southeast Greenland but slowly increased in the southwest, north, and northeast regions. Most outlet glaciers have been thinning during the last two decades, interrupted by episodes of decreasing thinning or even thickening. Dynamics of the major outlet glaciers dominated the mass loss from larger drainage basins, and simultaneous changes over distances up to 500 km are detected, indicating climate control. However, the intricate spatiotemporal pattern of dynamic thickness change suggests that, regardless of the forcing responsible for initial glacier acceleration and thinning, the response of individual glaciers is modulated by local conditions. Recent projections of dynamic contributions from the entire GrIS to SLR have been based on the extrapolation of four major outlet glaciers. Considering the observed complexity, we question how well these four glaciers represent all of Greenland's outlet glaciers.

  14. Partial structure factors reveal atomic dynamics in metallic alloy melts

    NASA Astrophysics Data System (ADS)

    Nowak, B.; Holland-Moritz, D.; Yang, F.; Voigtmann, Th.; Kordel, T.; Hansen, T. C.; Meyer, A.

    2017-07-01

    We investigate the dynamical decoupling of the diffusion coefficients of the different components in a metallic alloy melt, using a combination of neutron diffraction, isotopic substitution, and electrostatic levitation in Zr-Ni melts. We show that excess Ni atoms can diffuse more freely in a background of saturated chemical interaction, causing their dynamics to become much faster and thus decoupled than anticipated from the interparticle interactions. Based on the mode-coupling theory of the glass transition, the averaged structure as given by the partial static structure factors is able to explain the observed dynamical behavior.

  15. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    PubMed

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding.

  16. Quantitative phosphoproteomic analysis of porcine muscle within 24 h postmortem.

    PubMed

    Huang, Honggang; Larsen, Martin R; Palmisano, Giuseppe; Dai, Jie; Lametsch, René

    2014-06-25

    Protein phosphorylation can regulate most of the important processes in muscle, such as metabolism and contraction. The postmortem (PM) metabolism and rigor mortis have essential effects on meat quality. In order to identify and characterize the protein phosphorylation events involved in meat quality development, a quantitative mass spectrometry-based phosphoproteomic study was performed to analyze the porcine muscle within 24h PM using dimethyl labeling combined with the TiSH phosphopeptide enrichment strategy. In total 305 unique proteins were identified, including 160 phosphoproteins with 784 phosphorylation sites. Among these, 184 phosphorylation sites on 93 proteins had their phosphorylation levels significantly changed. The proteins involved in glucose metabolism and muscle contraction were the two largest clusters of phosphoproteins with significantly changed phosphorylation levels in muscle within 24 h PM. The high phosphorylation level of heat shock proteins (HSPs) in early PM may be an adaptive response to slaughter stress and protect muscle cell from apoptosis, as observed in the serine 84 of HSP27. This work indicated that PM muscle proteins underwent significant changes at the phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat quality development through the regulation of proteins involved in glucose metabolism and muscle contraction, thereby affecting glycolysis and rigor mortis development in PM muscle. The manuscript describes the characterization of postmortem (PM) porcine muscle within 24 h postmortem from the perspective of protein phosphorylation using advanced phosphoproteomic techniques. In the study, the authors employed the dimethyl labeling combined with the TiSH phosphopeptide enrichment and LC-MS/MS strategy. This was the first high-throughput quantitative phosphoproteomic study in PM muscle of farm animals. In the work, both the proteome

  17. Laser altimetry reveals complex pattern of Greenland Ice Sheet dynamics

    PubMed Central

    Csatho, Beata M.; Schenk, Anton F.; van der Veen, Cornelis J.; Babonis, Gregory; Duncan, Kyle; Rezvanbehbahani, Soroush; van den Broeke, Michiel R.; Simonsen, Sebastian B.; Nagarajan, Sudhagar; van Angelen, Jan H.

    2014-01-01

    We present a new record of ice thickness change, reconstructed at nearly 100,000 sites on the Greenland Ice Sheet (GrIS) from laser altimetry measurements spanning the period 1993–2012, partitioned into changes due to surface mass balance (SMB) and ice dynamics. We estimate a mean annual GrIS mass loss of 243 ± 18 Gt⋅y−1, equivalent to 0.68 mm⋅y−1 sea level rise (SLR) for 2003–2009. Dynamic thinning contributed 48%, with the largest rates occurring in 2004–2006, followed by a gradual decrease balanced by accelerating SMB loss. The spatial pattern of dynamic mass loss changed over this time as dynamic thinning rapidly decreased in southeast Greenland but slowly increased in the southwest, north, and northeast regions. Most outlet glaciers have been thinning during the last two decades, interrupted by episodes of decreasing thinning or even thickening. Dynamics of the major outlet glaciers dominated the mass loss from larger drainage basins, and simultaneous changes over distances up to 500 km are detected, indicating climate control. However, the intricate spatiotemporal pattern of dynamic thickness change suggests that, regardless of the forcing responsible for initial glacier acceleration and thinning, the response of individual glaciers is modulated by local conditions. Recent projections of dynamic contributions from the entire GrIS to SLR have been based on the extrapolation of four major outlet glaciers. Considering the observed complexity, we question how well these four glaciers represent all of Greenland’s outlet glaciers. PMID:25512537

  18. Reveal protein dynamics by combining computer simulation and neutron scattering

    NASA Astrophysics Data System (ADS)

    Hong, Liang; Smith, Jeremy; CenterMolecular Biophysics Team

    2014-03-01

    Protein carries out most functions in living things on the earth through characteristic modulation of its three-dimensional structure over time. Understanding the microscopic nature of the protein internal motion and its connection to the function and structure of the biomolecule is a central topic in biophysics, and of great practical importance for drug design, study of diseases, and the development of renewable energy, etc. Under physiological conditions, protein exhibits a complex dynamics landscape, i.e., a variety of diffusive and conformational motions occur on similar time and length scales. This variety renders difficult the derivation of a simplified description of protein internal motions in terms of a small number of distinct, additive components. This difficulty is overcome by our work using a combined approach of Molecular Dynamics (MD) simulations and the Neutron Scattering experiments. Our approach enables distinct protein motions to be characterized separately, furnishing an in-depth understanding of the connection between protein structure, dynamics and function.

  19. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research.

    PubMed

    Chan, Chi Yuet X'avia; Gritsenko, Marina A; Smith, Richard D; Qian, Wei-Jun

    2016-01-01

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.

  20. Structure and Dynamics of Four-way DNA Junctions Dynamics Revealed by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Lyubchenko, Yuri

    2004-03-01

    For-way DNA junctions (Holliday junctions) are critical intermediates for homologous, site-specific recombination, DNA repair and replication. A wealth of structural information is available for immobile four-way junctions. However, these data cannot give the answer on the mechanism of branch migration, the major property of the Holliday junction. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal, exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration. Note that it was the first time when the dynamics of the four-way DNA junction capable of branch migration had been analyzed. We applied time-lapse atomic force microscopy (single molecule dynamics AFM) to image directly loosely bound DNA at liquid-surface interface. These experiments show that mobile Holliday junctions adopt an unfolded conformation during branch migration. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. The analysis of the Holliday junctions dynamics at conditions limiting branch migration revealed a broad movement of the arms suggesting that the range of mobility of these junctions is much wider than detected before. Further applications of the time-lapse AFM approach in attempt to resolve the subpopulations of the junctions conformers and the prospects for analyses of dynamics of complex biological systems will be discussed.

  1. Rhodopsin Photoactivation Dynamics Revealed by Quasi-Elastic Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchhithranga M. C. D.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael; Chu, Xiang-Qiang

    2015-03-01

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision. During photoactivation, the chromophore retinal dissociates from protein yielding the opsin apoprotein. What are the changes in protein dynamics that occur during the photoactivation process? Here, we studied the microscopic dynamics of dark-state rhodopsin and the ligand-free opsin using quasielastic neutron scattering (QENS). The QENS technique tracks individual hydrogen atom motion because of the much higher neutron scattering cross-section of hydrogen than other atoms. We used protein with CHAPS detergent hydrated with heavy water. The activation of proteins is confirmed at low temperatures up to 300 K by mean-square displacement (MSD) analysis. The QENS experiments at temperatures ranging from 220 K to 300 K clearly indicate an increase in protein dynamic behavior with temperature. The relaxation time for the ligand-bound protein rhodopsin is faster compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which unlike protein, manifests localized motions.

  2. Communicability angles reveal critical edges for network consensus dynamics

    NASA Astrophysics Data System (ADS)

    Estrada, Ernesto; Vargas-Estrada, Eusebio; Ando, Hiroyasu

    2015-11-01

    We consider the question of determining how the topological structure influences a consensus dynamical processes taking place on a network. By considering a large data set of real-world networks we first determine that the removal of edges according to their communicability angle, an angle between position vectors of the nodes in an Euclidean communicability space, increases the average time of consensus by a factor of 5.68 in real-world networks. The edge betweenness centrality also identifies, in a smaller proportion, those critical edges for the consensus dynamics; i.e., its removal increases the time of consensus by a factor of 3.70 . We justify theoretically these findings on the basis of the role played by the algebraic connectivity and the isoperimetric number of networks on the dynamical process studied and their connections with the properties mentioned before. Finally, we study the role played by global topological parameters of networks on the consensus dynamics. We determine that the network density and the average distance-sum, which is analogous of the node degree for shortest-path distances, account for more than 80% of the variance of the average time of consensus in the real-world networks studied.

  3. Fire scars reveal variability and dynamics of eastern fire regimes

    Treesearch

    Richard P. Guyette; Daniel C. Dey; Michael C. Stambaugh; Rose-Marie Muzika

    2006-01-01

    Fire scar evidence in eastern North America is sparse and complex but shows promise in defining the dynamics of these fire regimes and their influence on ecosystems. We review fire scar data, methods, and limitations, and use this information to identify and examine the factors influencing fire regimes. Fire scar data from studies at more than 40 sites in Eastern North...

  4. Rhodopsin photoactivation dynamics revealed by quasi-elastic neutron scattering

    DOE PAGES

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchithranga M.d.c.; ...

    2015-01-27

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision under dim light conditions. During rhodopsin photoactivation, the chromophore retinal undergoes cis-trans isomerization, and subsequently dissociates from the protein yielding the opsin apoprotein [1]. What are the changes in protein dynamics that occur during the rhodopsin photoactivation process? Here, we studied the microscopic dynamics of the dark-state rhodopsin and the ligand-free opsin using quasi-elastic neutron scattering (QENS). The QENS technique tracks the individual hydrogen atom motions in the protein molecules, because the neutron scattering cross-section of hydrogen is much higher than other atoms [2-4]. We used protein (rhodopsin/opsin) samples with CHAPSmore » detergent hydrated with heavy water. The solvent signal is suppressed due to the heavy water, so that only the signals from proteins and detergents are detected. The activation of proteins is confirmed at low temperatures up to 300 K by the mean-square displacement (MSD) analysis. Our QENS experiments conducted at temperatures ranging from 220 K to 300 K clearly indicate that the protein dynamic behavior increases with temperature. The relaxation time for the ligand-bound protein rhodopsin was longer compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which forms a band around the protein molecule in the micelle. Unlike the protein, the CHAPS detergent manifests localized motions that are the same as in the bulk empty micelles. Furthermore QENS provides unique understanding of the key dynamics involved in the activation of the GPCR involved in the visual process.« less

  5. Rhodopsin photoactivation dynamics revealed by quasi-elastic neutron scattering

    SciTech Connect

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchithranga M.d.c.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael F.; Chu, Xiang -Qiang

    2015-01-27

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision under dim light conditions. During rhodopsin photoactivation, the chromophore retinal undergoes cis-trans isomerization, and subsequently dissociates from the protein yielding the opsin apoprotein [1]. What are the changes in protein dynamics that occur during the rhodopsin photoactivation process? Here, we studied the microscopic dynamics of the dark-state rhodopsin and the ligand-free opsin using quasi-elastic neutron scattering (QENS). The QENS technique tracks the individual hydrogen atom motions in the protein molecules, because the neutron scattering cross-section of hydrogen is much higher than other atoms [2-4]. We used protein (rhodopsin/opsin) samples with CHAPS detergent hydrated with heavy water. The solvent signal is suppressed due to the heavy water, so that only the signals from proteins and detergents are detected. The activation of proteins is confirmed at low temperatures up to 300 K by the mean-square displacement (MSD) analysis. Our QENS experiments conducted at temperatures ranging from 220 K to 300 K clearly indicate that the protein dynamic behavior increases with temperature. The relaxation time for the ligand-bound protein rhodopsin was longer compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which forms a band around the protein molecule in the micelle. Unlike the protein, the CHAPS detergent manifests localized motions that are the same as in the bulk empty micelles. Furthermore QENS provides unique understanding of the key dynamics involved in the activation of the GPCR involved in the visual process.

  6. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  7. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    PubMed

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here.

  8. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  9. Fluctuation power spectra reveal dynamical heterogeneity of peptides

    NASA Astrophysics Data System (ADS)

    Khatri, Bhavin; Yew, Zu Thur; Krivov, Sergei; McLeish, Tom; Paci, Emanuele

    2010-07-01

    Characterizing the conformational properties and dynamics of biopolymers and their relation to biological activity and function is an ongoing challenge. Single molecule techniques have provided a rich experimental window on these properties, yet they have often relied on simple one-dimensional projections of a multidimensional free energy landscape for a practical interpretation of the results. Here, we study three short peptides with different structural propensity (α helical, β hairpin, and random coil) in the presence (or absence) of a force applied to their ends using Langevin dynamics simulation and an all-atom model with implicit solvation. Each peptide produces fluctuation power spectra with a characteristic dynamic fingerprint consistent with persistent structural motifs of helices, hairpins, and random coils. The spectra for helix formation shows two well-defined relaxation modes, corresponding to local relaxation and cooperative coil to uncoil interconversion. In contrast, both the hairpin and random coil are polymerlike, showing a broad and continuous range of relaxation modes giving characteristic power laws of ω-5/4 and ω-3/2, respectively; the -5/4 power law for hairpins is robust and has not been previously observed. Langevin dynamics simulations of diffusers on a potential of mean force derived from the atomistic simulations fail to reproduce the fingerprints of each peptide motif in the power spectral density, demonstrating explicitly that such information is lacking in such one-dimensional projections. Our results demonstrate the yet unexploited potential of single molecule fluctuation spectroscopy to probe more fine scaled properties of proteins and biological macromolecules and how low dimensional projections may cause the loss of relevant information.

  10. Scanning angle interference microscopy reveals cell dynamics at the nanoscale.

    PubMed

    Paszek, Matthew J; DuFort, Christopher C; Rubashkin, Matthew G; Davidson, Michael W; Thorn, Kurt S; Liphardt, Jan T; Weaver, Valerie M

    2012-07-01

    Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.

  11. A Dynamical Model Reveals Gene Co-Localizations in Nucleus

    PubMed Central

    Yao, Ye; Lin, Wei; Hennessy, Conor; Fraser, Peter; Feng, Jianfeng

    2011-01-01

    Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclear region. A simple standard Brownian motion model in two and three dimensions shows that preferential co-localization is possible for co-regulated genes without any direct interaction, and suggests the occurrence may be due to a limitation in the number of available transcription factors. Experimental data of chromatin movements demonstrates that fractional rather than standard Brownian motion is more appropriate to model gene mobilizations, and we tested our dynamical model against recent static experimental data, using a sub-diffusion process by which the genes tend to colocalize more easily. Moreover, in order to compare our model with recently obtained experimental data, we studied the association level between genes and factors, and presented data supporting the validation of this dynamic model. As further applications of our model, we applied it to test against more biological observations. We found that increasing transcription factor number, rather than factory number and nucleus size, might be the reason for decreasing gene co-localization. In the scenario of frequency- or amplitude-modulation of transcription factors, our model predicted that frequency-modulation may increase the co-localization between its targeted genes. PMID:21760760

  12. Optogenetic perturbations reveal the dynamics of an oculomotor integrator

    PubMed Central

    Gonçalves, Pedro J.; Arrenberg, Aristides B.; Hablitzel, Bastian; Baier, Herwig; Machens, Christian K.

    2014-01-01

    Many neural systems can store short-term information in persistently firing neurons. Such persistent activity is believed to be maintained by recurrent feedback among neurons. This hypothesis has been fleshed out in detail for the oculomotor integrator (OI) for which the so-called “line attractor” network model can explain a large set of observations. Here we show that there is a plethora of such models, distinguished by the relative strength of recurrent excitation and inhibition. In each model, the firing rates of the neurons relax toward the persistent activity states. The dynamics of relaxation can be quite different, however, and depend on the levels of recurrent excitation and inhibition. To identify the correct model, we directly measure these relaxation dynamics by performing optogenetic perturbations in the OI of zebrafish expressing halorhodopsin or channelrhodopsin. We show that instantaneous, inhibitory stimulations of the OI lead to persistent, centripetal eye position changes ipsilateral to the stimulation. Excitatory stimulations similarly cause centripetal eye position changes, yet only contralateral to the stimulation. These results show that the dynamics of the OI are organized around a central attractor state—the null position of the eyes—which stabilizes the system against random perturbations. Our results pose new constraints on the circuit connectivity of the system and provide new insights into the mechanisms underlying persistent activity. PMID:24616666

  13. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

    PubMed Central

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  14. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    PubMed

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis.

    PubMed

    Grimaud, Florent; Rogniaux, Hélène; James, Martha G; Myers, Alan M; Planchot, Véronique

    2008-01-01

    In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein-protein interactions.

  16. Dynamic Monitoring Reveals Motor Task Characteristics in Prehistoric Technical Gestures

    PubMed Central

    Pfleging, Johannes; Stücheli, Marius; Iovita, Radu; Buchli, Jonas

    2015-01-01

    Reconstructing ancient technical gestures associated with simple tool actions is crucial for understanding the co-evolution of the human forelimb and its associated control-related cognitive functions on the one hand, and of the human technological arsenal on the other hand. Although the topic of gesture is an old one in Paleolithic archaeology and in anthropology in general, very few studies have taken advantage of the new technologies from the science of kinematics in order to improve replicative experimental protocols. Recent work in paleoanthropology has shown the potential of monitored replicative experiments to reconstruct tool-use-related motions through the study of fossil bones, but so far comparatively little has been done to examine the dynamics of the tool itself. In this paper, we demonstrate that we can statistically differentiate gestures used in a simple scraping task through dynamic monitoring. Dynamics combines kinematics (position, orientation, and speed) with contact mechanical parameters (force and torque). Taken together, these parameters are important because they play a role in the formation of a visible archaeological signature, use-wear. We present our new affordable, yet precise methodology for measuring the dynamics of a simple hide-scraping task, carried out using a pull-to (PT) and a push-away (PA) gesture. A strain gage force sensor combined with a visual tag tracking system records force, torque, as well as position and orientation of hafted flint stone tools. The set-up allows switching between two tool configurations, one with distal and the other one with perpendicular hafting of the scrapers, to allow for ethnographically plausible reconstructions. The data show statistically significant differences between the two gestures: scraping away from the body (PA) generates higher shearing forces, but requires greater hand torque. Moreover, most benchmarks associated with the PA gesture are more highly variable than in the PT gesture

  17. Dynamic Monitoring Reveals Motor Task Characteristics in Prehistoric Technical Gestures.

    PubMed

    Pfleging, Johannes; Stücheli, Marius; Iovita, Radu; Buchli, Jonas

    2015-01-01

    Reconstructing ancient technical gestures associated with simple tool actions is crucial for understanding the co-evolution of the human forelimb and its associated control-related cognitive functions on the one hand, and of the human technological arsenal on the other hand. Although the topic of gesture is an old one in Paleolithic archaeology and in anthropology in general, very few studies have taken advantage of the new technologies from the science of kinematics in order to improve replicative experimental protocols. Recent work in paleoanthropology has shown the potential of monitored replicative experiments to reconstruct tool-use-related motions through the study of fossil bones, but so far comparatively little has been done to examine the dynamics of the tool itself. In this paper, we demonstrate that we can statistically differentiate gestures used in a simple scraping task through dynamic monitoring. Dynamics combines kinematics (position, orientation, and speed) with contact mechanical parameters (force and torque). Taken together, these parameters are important because they play a role in the formation of a visible archaeological signature, use-wear. We present our new affordable, yet precise methodology for measuring the dynamics of a simple hide-scraping task, carried out using a pull-to (PT) and a push-away (PA) gesture. A strain gage force sensor combined with a visual tag tracking system records force, torque, as well as position and orientation of hafted flint stone tools. The set-up allows switching between two tool configurations, one with distal and the other one with perpendicular hafting of the scrapers, to allow for ethnographically plausible reconstructions. The data show statistically significant differences between the two gestures: scraping away from the body (PA) generates higher shearing forces, but requires greater hand torque. Moreover, most benchmarks associated with the PA gesture are more highly variable than in the PT gesture

  18. Dynamics of the Wulong Landslide Revealed by Broadband Seismic Records

    NASA Astrophysics Data System (ADS)

    Huang, X.; Dan, Y.

    2016-12-01

    Long-period seismic signals are frequently used to trace the dynamic process of large scale landslides. The catastrophic WuLong landslide occurred at 14:51 on 5 June 2009 (Beijing time, UTC+8) in Wulong Prefecture, Southwest China. The topography in landslide area varies dramatically, enhancing the complexity in its movement characteristics. The mass started sliding northward on the upper part of the cliff located upon the west slope of the Tiejianggou gully, and shifted its movement direction to northeastward after being blocked by stable bedrock in front, leaving a scratch zone. The sliding mass then moved downward along the west slope of the gully until it collided with the east slope, and broke up into small pieces after the collision, forming a debris flow along the gully. We use long-period seismic signals extracted from eight broadband seismic stations within 250 km of the landslide to estimate its source time functions. Combining with topographic surveys done before and after the event, we can also resolve kinematic parameters of sliding mass, i.e. velocities, displacements and trajectories, perfectly characterizing its movement features. The runout trajectory deduced from source time functions is consistent with the sliding path, including two direction changing processes, corresponding to scratching the western bedrock and collision with the east slope respectively. Topographic variations can be reflected from estimated velocities. The maximum velocity of the sliding mass reaches 35 m/s before the collision with the east slope of the Tiejianggou gully, resulting from the height difference between the source zone and the deposition zone. What is important is that dynamics of scratching and collision can be characterized by source time functions. Our results confirm that long-period seismic signals are sufficient to characterize dynamics and kinematics of large scale landslides which occur in a region with complex topography.

  19. Differential DNase I hypersensitivity reveals factor-dependent chromatin dynamics

    PubMed Central

    He, Housheng Hansen; Meyer, Clifford A.; Chen, Mei Wei; Brown, Myles; Liu, X. Shirley

    2012-01-01

    Transcription factor cistromes are highly cell-type specific. Chromatin accessibility, histone modifications, and nucleosome occupancy have all been found to play a role in defining these binding locations. Here, we show that hormone-induced DNase I hypersensitivity changes (ΔDHS) are highly predictive of androgen receptor (AR) and estrogen receptor 1 (ESR1) binding in prostate cancer and breast cancer cells, respectively. While chromatin structure prior to receptor binding and nucleosome occupancy after binding are strikingly different for ESR1 and AR, ΔDHS is highly predictive for both. AR binding is associated with changes in both local nucleosome occupancy and DNase I hypersensitivity. In contrast, while global ESR1 binding is unrelated to changes in nucleosome occupancy, DNase I hypersensitivity dynamics are also predictive of the ESR1 cistrome. These findings suggest that AR and ESR1 have distinct modes of interaction with chromatin and that DNase I hypersensitivity dynamics provides a general approach for predicting cell-type specific cistromes. PMID:22508765

  20. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    NASA Astrophysics Data System (ADS)

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; Urban, Volker S.; Pakrasi, Himadri B.; Ohl, Michael

    2016-01-01

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  1. Photon echo spectroscopy reveals structure-dynamics relationships in carotenoids

    NASA Astrophysics Data System (ADS)

    Christensson, N.; Polivka, T.; Yartsev, A.; Pullerits, T.

    2009-06-01

    Based on simultaneous analysis of the frequency-resolved transient grating, peak shift, and echo width signals, we present a model for the third-order optical response of carotenoids including population dynamics and system-bath interactions. Our frequency-resolved photon echo experiments show that the model needs to incorporate the excited-state absorption from both the S2 and the S1 states. We apply our model to analyze the experimental results on astaxanthin and lycopene, aiming to elucidate the relation between structure and system-bath interactions. Our analysis allows us to relate structural motifs to changes in the energy-gap correlation functions. We find that the terminal rings of astaxanthin lead to increased coupling between slow molecular motions and the electronic transition. We also find evidence for stronger coupling to higher frequency overdamped modes in astaxanthin, pointing to the importance of the functional groups in providing coupling to fluctuations influencing the dynamics in the passage through the conical intersection governing the S2-S1 relaxation.

  2. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    DOE PAGES

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Liberton, Michelle; ...

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. Here, we present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity.more » High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. Moreover, we observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Finally, our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.« less

  3. Genomic analysis of regulatory network dynamics reveals large topological changes

    NASA Astrophysics Data System (ADS)

    Luscombe, Nicholas M.; Madan Babu, M.; Yu, Haiyuan; Snyder, Michael; Teichmann, Sarah A.; Gerstein, Mark

    2004-09-01

    Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here-particularly the large-scale topological changes and hub transience-will apply to other biological networks, including complex sub-systems in higher eukaryotes.

  4. GRACE storage-runoff hystereses reveal the dynamics of ...

    EPA Pesticide Factsheets

    Watersheds function as integrated systems where climate and geology govern the movement of water. In situ instrumentation can provide local-scale insights into the non-linear relationship between streamflow and water stored in a watershed as snow, soil moisture, and groundwater. However, there is a poor understanding of these processes at the regional scale—primarily because of our inability to measure water stores and fluxes in the subsurface. Now NASA’s Gravity Recovery and Climate Experiment (GRACE) satellites quantify changes in the amount of water stored across and through the Earth, providing measurements of regional hydrologic behavior. Here we apply GRACE data to characterize for the first time how regional watersheds function as simple, dynamic systems through a series of hysteresis loops. While the physical processes underlying the loops are inherently complex, the vertical integration of terrestrial water in the GRACE signal provides process-based insights into the dynamic and non-linear function of regional-scale watersheds. We use this process-based understanding with GRACE data to effectively forecast seasonal runoff (mean R2 of 0.91) and monthly runoff (mean R2 of 0.77) in three regional-scale watersheds (>150,000 km2) of the Columbia River Basin, USA. Data from the Gravity Recovery and Climate Experiment (GRACE) satellites provide a novel dataset for understanding changes in the amount of water stored across and through the surface of the Ear

  5. Revealing the Dynamics of Thylakoid Membranes in Living Cyanobacterial Cells

    SciTech Connect

    Stingaciu, Laura-Roxana; O’Neill, Hugh; Urban, Volker S.; Ohl, Michael

    2016-01-21

    Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. Here, we present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. Moreover, we observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Finally, our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.

  6. Genomic analysis of regulatory network dynamics reveals large topological changes.

    PubMed

    Luscombe, Nicholas M; Babu, M Madan; Yu, Haiyuan; Snyder, Michael; Teichmann, Sarah A; Gerstein, Mark

    2004-09-16

    Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here--particularly the large-scale topological changes and hub transience--will apply to other biological networks, including complex sub-systems in higher eukaryotes.

  7. Invisible Electronic States and Their Dynamics Revealed by Perturbations

    NASA Astrophysics Data System (ADS)

    Merer, Anthony J.

    2011-06-01

    Sooner or later everyone working in the field of spectroscopy encounters perturbations. These can range in size from a small shift of a single rotational level to total destruction of the vibrational and rotational patterns of an electronic state. To some workers perturbations are a source of terror, but to others they are the most fascinating features of molecular spectra, because they give information about molecular dynamics, and about states that would otherwise be invisible as a result of unfavorable selection rules. An example of the latter is the essentially complete characterization of the tilde{b}^3A_2 state of SO_2 from the vibronic perturbations it causes in the tilde{a}^3B_1 state. The S_1-trans state of acetylene is a beautiful example of dynamics in action. The level patterns of the three bending vibrations change dramatically with increasing vibrational excitation as a result of the vibrational angular momentum and the approach to the isomerization barrier. Several vibrational levels of the S_1-cis isomer, previously thought to be unobservable, can now be assigned. They obtain their intensity through interactions with nearby levels of the trans isomer.

  8. Visual analysis and dynamical control of phosphoproteomic networks

    NASA Astrophysics Data System (ADS)

    Meyer-Bäse, Anke; Görke, Robert; Lobbes, Marc; Emmett, Mark R.; Nilsson, Carol L.

    2013-05-01

    This paper presents novel graph algorithms and modern control solutions applied to the graph networks resulting from specific experiments to discover disease-related pathways and drug targets in glioma cancer stem cells (GSCs). The theoretical framework applies to many other high-throughput data from experiments relevant to a variety of diseases. In addition to developing novel graph and control networks to predict therapeutic targets, these algorithms will provide biochemists with techniques to identify more metabolic regions and biological pathways for complex diseases, and design and test novel therapeutic solutions.

  9. Charge-dependent conformations and dynamics of pamam dendrimers revealed by neutron scattering and molecular dynamics

    NASA Astrophysics Data System (ADS)

    Wu, Bin

    spatial instrumental scales, understanding experimental results involves extensive and difficult data analysis based on liquid theory and condensed matter physics. Therefore, a model that successfully describes the inter- and intra-dendrimer correlations is crucial in obtaining and delivering reliable information. On the other hand, making meaningful comparisons between molecular dynamics and neutron scattering is a fundamental challenge to link simulations and experiments at the nano-scale. This challenge stems from our approach to utilize MD simulation to explain the underlying mechanism of experimental observation. The SANS measurements were conducted on a series of SANS spectrometers including the Extended Q-Range Small-Angle Neutron Scattering Diffractometer (EQ-SANS) and the General-Purpose Small-Angle Neutron Scattering Diffractometer (GP-SANS) at the Oak Ridge National Laboratory (ORNL), and NG7 Small Angle Neutron Scattering Spectrometer at National Institute of Standards (NIST) and Technology in U.S.A., large dynamic range small-angle diffractometer D22 at Institut Laue-Langevin (ILL) in France, and 40m-SANS Spectrometer at Korea Atomic Energy Research Institute (KAERI) in Korea. On the other hand, the Amber molecular dynamics simulation package is utilized to carry out the computational study. In this dissertation, the following observations have been revealed. The previously developed theoretical model for polyelectrolyte dendrimers are adopted to analyze SANS measurements and superb model fitting quality is found. Coupling with advanced contrast variation small angle neutron scattering (CVSANS) data analysis scheme reported recently, the intra-dendrimer hydration and hydrocarbon components distributions are revealed experimentally. The results indeed indicate that the maximum density is located in the molecular center rather than periphery, which is consistent to previous SANS studies and the back-folding picture of PAMAM dendrimers. According to this picture

  10. Shapiro like steps reveals molecular nanomagnets’ spin dynamics

    SciTech Connect

    Abdollahipour, Babak; Abouie, Jahanfar Ebrahimi, Navid

    2015-09-15

    We present an accurate way to detect spin dynamics of a nutating molecular nanomagnet by inserting it in a tunnel Josephson junction and studying the current voltage (I-V) characteristic. The spin nutation of the molecular nanomagnet is generated by applying two circularly polarized magnetic fields. We demonstrate that modulation of the Josephson current by the nutation of the molecular nanomagnet’s spin appears as a stepwise structure like Shapiro steps in the I-V characteristic of the junction. Width and heights of these Shapiro-like steps are determined by two parameters of the spin nutation, frequency and amplitude of the nutation, which are simply tuned by the applied magnetic fields.

  11. Revealing the dynamics of polymicrobial infections: implications for antibiotic therapy

    PubMed Central

    Rogers, Geraint B.; Hoffman, Lucas R.; Whiteley, Marvin; Daniels, Thomas W.V.; Carroll, Mary P.; Bruce, Kenneth D.

    2011-01-01

    As a new generation of culture-independent analytical strategies emerge, the amount of data on polymicrobial infections will increase dramatically. For these data to inform clinical thinking, and in turn to maximise benefits for patients, an appropriate framework for their interpretation is required. Here, we use cystic fibrosis (CF) lower airway infections as a model system to examine how conceptual and technological advances can address two clinical questions that are central to improved management of CF respiratory disease. Firstly, can markers of the microbial community be identified that predict a change in infection dynamics and clinical outcomes? Secondly, can these new strategies directly characterize the impact of antimicrobial therapies, allowing treatment efficacy to be both assessed and optimized? PMID:20554204

  12. Bacterial associations reveal spatial population dynamics in Anopheles gambiae mosquitoes

    PubMed Central

    Buck, Moritz; Nilsson, Louise K. J.; Brunius, Carl; Dabiré, Roch K.; Hopkins, Richard; Terenius, Olle

    2016-01-01

    The intolerable burden of malaria has for too long plagued humanity and the prospect of eradicating malaria is an optimistic, but reachable, target in the 21st century. However, extensive knowledge is needed about the spatial structure of mosquito populations in order to develop effective interventions against malaria transmission. We hypothesized that the microbiota associated with a mosquito reflects acquisition of bacteria in different environments. By analyzing the whole-body bacterial flora of An. gambiae mosquitoes from Burkina Faso by 16 S amplicon sequencing, we found that the different environments gave each mosquito a specific bacterial profile. In addition, the bacterial profiles provided precise and predicting information on the spatial dynamics of the mosquito population as a whole and showed that the mosquitoes formed clear local populations within a meta-population network. We believe that using microbiotas as proxies for population structures will greatly aid improving the performance of vector interventions around the world. PMID:26960555

  13. Epitope flexibility and dynamic footprint revealed by molecular dynamics of a pMHC-TCR complex.

    PubMed

    Reboul, Cyril F; Meyer, Grischa R; Porebski, Benjamin T; Borg, Natalie A; Buckle, Ashley M

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

  14. Epitope Flexibility and Dynamic Footprint Revealed by Molecular Dynamics of a pMHC-TCR Complex

    PubMed Central

    Porebski, Benjamin T.; Borg, Natalie A.; Buckle, Ashley M.

    2012-01-01

    The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent ‘snapshots’ and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6–9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into ‘scanning’ mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography. PMID:22412359

  15. The complexity of gene expression dynamics revealed by permutation entropy

    PubMed Central

    2010-01-01

    Background High complexity is considered a hallmark of living systems. Here we investigate the complexity of temporal gene expression patterns using the concept of Permutation Entropy (PE) first introduced in dynamical systems theory. The analysis of gene expression data has so far focused primarily on the identification of differentially expressed genes, or on the elucidation of pathway and regulatory relationships. We aim to study gene expression time series data from the viewpoint of complexity. Results Applying the PE complexity metric to abiotic stress response time series data in Arabidopsis thaliana, genes involved in stress response and signaling were found to be associated with the highest complexity not only under stress, but surprisingly, also under reference, non-stress conditions. Genes with house-keeping functions exhibited lower PE complexity. Compared to reference conditions, the PE of temporal gene expression patterns generally increased upon stress exposure. High-complexity genes were found to have longer upstream intergenic regions and more cis-regulatory motifs in their promoter regions indicative of a more complex regulatory apparatus needed to orchestrate their expression, and to be associated with higher correlation network connectivity degree. Arabidopsis genes also present in other plant species were observed to exhibit decreased PE complexity compared to Arabidopsis specific genes. Conclusions We show that Permutation Entropy is a simple yet robust and powerful approach to identify temporal gene expression profiles of varying complexity that is equally applicable to other types of molecular profile data. PMID:21176199

  16. Indole Localization in an Explicit Bilayer Revealed via Molecular Dynamics

    NASA Astrophysics Data System (ADS)

    Norman, Kristen

    2005-11-01

    It is well known that the amino-acid tryptophan is particularly stable in the interfacial region of biological membranes, and this preference is a property of the tryptophan side-chain. Analogues of this side-chain, such as indole, strongly localize in the interfacial region, especially near the glycerol moiety of the lipids in the bilayer. Using molecular dynamics calculations, we determine the potential of mean force (PMF) for indoles in the bilayer. We compare the calculated PMF for indole with that of benzene to show that exclusion from the center of the lipid bilayer does not occur in all aromatics, but is strong in indoles. We find three minima in the PMF. Indole is most stabilized near the glycerol moiety. A weaker binding location is found near the choline groups of the lipid molecules. An even weaker binding side is found near the center of the lipid hydrocarbon core. Comparisions between uncharged, weakly charged, and highly charged indoles demonstrate that the exclusion is caused by the charge distribution on the indole rather than the ``lipo-phobic'' effect. High temperature simulations are used to determine the relative contribution of enthalpy and entropy to indole localization. The orientation of indole is found to be largely charge independent and is a strong function of depth within the bilayer. We find good agreement between simulated SCD order parameters for indole and experimentally determined order parameters.

  17. Dynamic Zebrafish Interactome Reveals Transcriptional Mechanisms of Dioxin Toxicity

    PubMed Central

    Alexeyenko, Andrey; Wassenberg, Deena M.; Lobenhofer, Edward K.; Yen, Jerry; Linney, Elwood; Sonnhammer, Erik L. L.; Meyer, Joel N.

    2010-01-01

    Background In order to generate hypotheses regarding the mechanisms by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) causes toxicity, we analyzed global gene expression changes in developing zebrafish embryos exposed to this potent toxicant in the context of a dynamic gene network. For this purpose, we also computationally inferred a zebrafish (Danio rerio) interactome based on orthologs and interaction data from other eukaryotes. Methodology/Principal Findings Using novel computational tools to analyze this interactome, we distinguished between dioxin-dependent and dioxin-independent interactions between proteins, and tracked the temporal propagation of dioxin-dependent transcriptional changes from a few genes that were altered initially, to large groups of biologically coherent genes at later times. The most notable processes altered at later developmental stages were calcium and iron metabolism, embryonic morphogenesis including neuronal and retinal development, a variety of mitochondria-related functions, and generalized stress response (not including induction of antioxidant genes). Within the interactome, many of these responses were connected to cytochrome P4501A (cyp1a) as well as other genes that were dioxin-regulated one day after exposure. This suggests that cyp1a may play a key role initiating the toxic dysregulation of those processes, rather than serving simply as a passive marker of dioxin exposure, as suggested by earlier research. Conclusions/Significance Thus, a powerful microarray experiment coupled with a flexible interactome and multi-pronged interactome tools (which are now made publicly available for microarray analysis and related work) suggest the hypothesis that dioxin, best known in fish as a potent cardioteratogen, has many other targets. Many of these types of toxicity have been observed in mammalian species and are potentially caused by alterations to cyp1a. PMID:20463971

  18. Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis.

    PubMed

    Gahloth, Deepankar; Dunstan, Mark S; Quaglia, Daniela; Klumbys, Evaldas; Lockhart-Cairns, Michael P; Hill, Andrew M; Derrington, Sasha R; Scrutton, Nigel S; Turner, Nicholas J; Leys, David

    2017-09-01

    Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.

  19. Timetree of Aselloidea reveals species diversification dynamics in groundwater.

    PubMed

    Morvan, Claire; Malard, Florian; Paradis, Emmanuel; Lefébure, Tristan; Konecny-Dupré, Lara; Douady, Christophe J

    2013-07-01

    A key challenge for biologists is to document and explain global patterns of diversification in a wide range of environments. Here, we explore patterns of continental-scale diversification in a groundwater species-rich clade, the superfamily Aselloidea (Pancrustacea: Isopoda). Our analyses supported a constant diversification rate during most of the course of Aselloidea evolution, until 4-15 Ma when diversification rates started to decrease. This constant accumulation of lineages challenges the view that groundwater species diversification in temperate regions might have been primarily driven by major changes in physical environment leading to the extinction of surface populations and subsequent synchronous isolation of multiple groundwater populations. Rather than acting synchronously over broad geographic regions, factors causing extinction of surface populations and subsequent reproductive isolation of groundwater populations may act in a local and asynchronous manner, thereby resulting in a constant speciation rate over time. Our phylogeny also revealed several cases of parapatric distributions among closely related surface-water and groundwater species suggesting that species diversification could also arise from a process of disruptive selection along the surface-subterranean environmental gradient. Our results call for re-evaluating the spatial scale and timing of factors causing diversification events in groundwater.

  20. Dynamical analysis reveals individuality of locomotion in goldfish.

    PubMed

    Neumeister, H; Cellucci, C J; Rapp, P E; Korn, H; Faber, D S

    2004-02-01

    Goldfish swimming was analysed quantitatively to determine if it exhibits distinctive individual spatio-temporal patterns. Due to the inherent variability in fish locomotion, this hypothesis was tested using five nonlinear measures, complemented by mean velocity. A library was constructed of 75 trajectories, each of 5 min duration, acquired from five fish swimming in a constant and relatively homogeneous environment. Three nonlinear measures, the 'characteristic fractal dimension' and 'Richardson dimension', both quantifying the degree to which a trajectory departs from a straight line, and 'relative dispersion', characterizing the variance as a function of the duration, have coefficients of variation less than 7%, in contrast to mean velocity (30%). A discriminant analysis, or classification system, based on all six measures revealed that trajectories are indeed highly individualistic, with the probability that any two trajectories generated from different fish are equivalent being less than 1%. That is, the combination of these measures allows a given trajectory to be assigned to its source with a high degree of confidence. The Richardson dimension and the 'Hurst exponent', which quantifies persistence, were the most effective measures.

  1. Persistent predator–prey dynamics revealed by mass extinction

    PubMed Central

    Sallan, Lauren Cole; Kammer, Thomas W.; Ausich, William I.; Cook, Lewis A.

    2011-01-01

    Predator–prey interactions are thought by many researchers to define both modern ecosystems and past macroevolutionary events. In modern ecosystems, experimental removal or addition of taxa is often used to determine trophic relationships and predator identity. Both characteristics are notoriously difficult to infer in the fossil record, where evidence of predation is usually limited to damage from failed attacks, individual stomach contents, one-sided escalation, or modern analogs. As a result, the role of predation in macroevolution is often dismissed in favor of competition and abiotic factors. Here we show that the end-Devonian Hangenberg event (359 Mya) was a natural experiment in which vertebrate predators were both removed and added to an otherwise stable prey fauna, revealing specific and persistent trophic interactions. Despite apparently favorable environmental conditions, crinoids diversified only after removal of their vertebrate consumers, exhibiting predatory release on a geological time scale. In contrast, later Mississippian (359–318 Mya) camerate crinoids declined precipitously in the face of increasing predation pressure from new durophagous fishes. Camerate failure is linked to the retention of obsolete defenses or “legacy adaptations” that prevented coevolutionary escalation. Our results suggest that major crinoid evolutionary phenomena, including rapid diversification, faunal turnover, and species selection, might be linked to vertebrate predation. Thus, interactions observed in small ecosystems, such as Lotka-Volterra cycles and trophic cascades, could operate at geologic time scales and higher taxonomic ranks. Both trophic knock-on effects and retention of obsolete traits might be common in the aftermath of predator extinction. PMID:21536875

  2. Oman metamorphic sole formation reveals early subduction dynamics

    NASA Astrophysics Data System (ADS)

    Soret, Mathieu; Agard, Philippe; Dubacq, Benoît; Plunder, Alexis; Ildefonse, Benoît; Yamato, Philippe; Prigent, Cécile

    2016-04-01

    Metamorphic soles correspond to m to ~500m thick tectonic slices welded beneath most of the large-scale ophiolites. They typically show a steep inverted metamorphic structure where the pressure and temperature conditions of crystallization increase upward (from 500±100°C at 0.5±0.2 GPa to 800±100°C at 1.0±0.2 GPa), with isograds subparallel to the contact with the overlying ophiolitic peridotite. The proportion of mafic rocks in metamorphic soles also increases from the bottom (meta-sediments rich) to the top (approaching the ophiolite peridotites). These soles are interpreted as the result of heat transfer from the incipient mantle wedge toward the nascent slab (associated with large-scale fluid transfer and possible shear heating) during the first My of intra-oceanic subduction (as indicated by radiometric ages). Metamorphic soles provide therefore major constraints on early subduction dynamics (i.e., thermal structure, fluid migration and rheology along the nascent slab interface). We present a detailed structural and petrological study of the metamorphic sole from 4 major cross-sections along the Oman ophiolite. We show precise pressure-temperature estimates obtained by pseudosection modelling and EBSD measurements performed on both the garnet-bearing and garnet-free high-grade sole. Results allow quantification of the micro-scale deformation and highlight differences in pressure-temperature-deformation conditions between the 4 different locations, showing that the inverted metamorphic gradient through the sole is not continuous in all locations. Based on these new constraints, we suggest a new tectonic-petrological model for the formation of metamorphic soles below ophiolites. This model involves the stacking of several homogeneous slivers of oceanic crust leading to the present-day structure of the sole. In this view, these thrusts are the result of rheological contrasts between the sole and the peridotite as the plate interface progressively cools down

  3. Quantitative phosphoproteomics by mass spectrometry: Past, present, and future

    PubMed Central

    Nita-Lazar, Aleksandra; Saito-Benz, Hideshiro; White, Forest M.

    2009-01-01

    Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight. PMID:18846511

  4. HUBBLE IMAGES REVEAL A YOUNG STAR'S DYNAMIC DISK AND JETS

    NASA Technical Reports Server (NTRS)

    2002-01-01

    These images of HH 30 show changes over only a five-year period in the disk and jets of this newborn star, which is about half a million years old. The pictures were taken between 1995 and 2000 with the Wide Field and Planetary Camera 2 aboard NASA's Hubble Space Telescope. Astronomers are interested in the disk because it is probably similar to the one from which the Sun and the planets in our solar system formed. Hubble reveals an edge-on disk (located at the bottom of the images), which appears as a flattened cloud of dust split into two halves by a dark lane. The disk blocks light from the central star. All that is visible is the reflection of the star's light by dust above and below the plane of the disk. The disk's diameter is 450 astronomical units (one astronomical unit equals the Earth-Sun distance). Shadows billions of miles in size can be seen moving across the disk. In 1995 and 2000, the left and right sides of the disk were about the same brightness, but in 1998 the right side was brighter. These patterns may be caused by bright spots on the star or variations in the disk near the star. The dust cloud near the top of these frames is illuminated by the star and reflects changes in its brightness. The star's magnetic field plays a major role in forming the jets (located above and below the disk), which look like streams of water from a fire hose. The powerful magnetic field creates the jets by channeling gas from the disk along the magnetic poles above and below the star. The gaps between the compact knots of gas seen in the jet above the disk indicate that this is a sporadic process. By tracking the motion of these knots over time, astronomers have measured the jet's speed at between 200,000 to 600,000 miles per hour (160,000 and 960,000 kilometers per hour). Oddly, the jet below the disk is moving twice as fast as the one above it. Credits: NASA, Alan Watson (Universidad Nacional Autonoma de Mexico), Karl Stapelfeldt (Jet Propulsion Laboratory), John

  5. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    PubMed

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or < 0.5) in response to spontaneous or oxytocin-stimulated contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P < 0.05) and oxytocin -induced (18.56 ± 8.18 fold, P < 0.01) contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  6. Self-similar multiscale structure of lignin revealed by neutron scattering and molecular dynamics simulation

    SciTech Connect

    Petridis, Loukas; Pingali, Sai Venkatesh; Urban, Volker; Heller, William T; O'Neill, Hugh Michael; Foston, Marcus B; Ragauskas, Arthur J; Smith, Jeremy C

    2011-01-01

    Lignin, a major polymeric component of plant cell walls, forms aggregates in vivo and poses a barrier to cellulosic ethanol production. Here, neutron scattering experiments and molecular dynamics simulations reveal that lignin aggregates are characterized by a surface fractal dimension that is invariant under change of scale from 1 1000 A. The simulations also reveal extensive water penetration of the aggregates and heterogeneous chain dynamics corresponding to a rigid core with a fluid surface.

  7. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro *

    PubMed Central

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-01-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

  8. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    PubMed

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

  9. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  10. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    PubMed

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-07

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

  11. Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

    PubMed

    Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

    2015-01-01

    In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins

  12. Slow dynamics of nanocomposite polymer aerogels as revealed by X-ray photocorrelation spectroscopy (XPCS).

    PubMed

    Hernández, Rebeca; Nogales, Aurora; Sprung, Michael; Mijangos, Carmen; Ezquerra, Tiberio A

    2014-01-14

    We report on a novel slow dynamics of polymer xerogels, aerogels, and nanocomposite aerogels with iron oxide nanoparticles, as revealed by X-ray photon correlation spectroscopy. The polymer aerogel and its nanocomposite aerogels, which are porous in nature, exhibit hyper-diffusive dynamics at room temperature. In contrast, non-porous polymer xerogels exhibit an absence of this peculiar dynamics. This slow dynamical process has been assigned to a relaxation of the characteristic porous structure of these materials and not to the presence of nanoparticles.

  13. Slow dynamics of nanocomposite polymer aerogels as revealed by X-ray photocorrelation spectroscopy (XPCS)

    SciTech Connect

    Hernández, Rebeca E-mail: aurora.nogales@csic.es; Mijangos, Carmen; Nogales, Aurora E-mail: aurora.nogales@csic.es; Ezquerra, Tiberio A.; Sprung, Michael

    2014-01-14

    We report on a novel slow dynamics of polymer xerogels, aerogels, and nanocomposite aerogels with iron oxide nanoparticles, as revealed by X-ray photon correlation spectroscopy. The polymer aerogel and its nanocomposite aerogels, which are porous in nature, exhibit hyper-diffusive dynamics at room temperature. In contrast, non-porous polymer xerogels exhibit an absence of this peculiar dynamics. This slow dynamical process has been assigned to a relaxation of the characteristic porous structure of these materials and not to the presence of nanoparticles.

  14. Revealing radiotherapy- and chemoradiation-induced pathway dynamics in glioblastoma by analyzing multiple differential networks.

    PubMed

    Zhou, Jia; Chen, Chao; Li, Hua-Feng; Hu, Yu-Jie; Xie, Hong-Ling

    2017-07-01

    The progression of glioblastoma (GBM) is driven by dynamic alterations in the activity and connectivity of gene pathways. Revealing these dynamic events is necessary in order to understand the pathological mechanisms of, and develop effective treatments for, GBM. The present study aimed to investigate dynamic alterations in pathway activity and connectivity across radiotherapy and chemoradiation conditions in GBM, and to give system‑level insights into molecular mechanisms for GBM therapy. A total of two differential co‑expression networks (DCNs) were constructed using Pearson correlation coefficient analysis and one sided t‑tests, based on gene expression profiles and protein‑protein interaction networks, one for each condition. Subsequently, shared differential modules across DCNs were detected via significance analysis for candidate modules, which were obtained according to seed selection, module search by seed expansion and refinement of searched modules. As condition‑specific differential modules mediate differential biological processes, the module connectivity dynamic score (MCDS) was implemented to explore dynamic alterations among them. Based on DCNs with 287 nodes and 1,052 edges, a total of 28 seed genes and seven candidate modules were identified. Following significance analysis, five shared differential modules were identified in total. Dynamic alterations among these differential modules were identified using the MCDS, and one module with significant dynamic alterations was identified, termed the dynamic module. The present study revealed the dynamic alterations of shared differential modules, identified one dynamic module between the radiotherapy and chemoradiation conditions, and demonstrated that pathway dynamics may applied to the study of the pathogenesis and therapy of GBM.

  15. Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

    PubMed

    Islam, Md Shariful; Nolte, Hendrik; Jacob, Wright; Ziegler, Anna B; Pütz, Stefanie; Grosjean, Yael; Szczepanowska, Karolina; Trifunovic, Aleksandra; Braun, Thomas; Heumann, Hermann; Heumann, Rolf; Hovemann, Bernhard; Moore, Darren J; Krüger, Marcus

    2016-10-29

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson's disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

  16. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

    PubMed

    Soderblom, Erik J; Philipp, Melanie; Thompson, J Will; Caron, Marc G; Moseley, M Arthur

    2011-05-15

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

  17. Cross-Species PTM Mapping from Phosphoproteomic Data.

    PubMed

    Chaudhuri, Rima; Yang, Jean Yee Hwa

    2017-01-01

    Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data. Cross-species comparison of PTM sites is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on previously annotated orthologous phosphosites and do not enable cross-species mapping of newly identified modification sites. Here, we describe an automated web-based tool, PhosphOrtholog, that accurately maps annotated and novel orthologous PTM sites from high-throughput MS-based experimental data obtained from different species without relying on existing PTM databases. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of evolutionarily conserved and functional PTM sites that influence most biological processes. In this Chapter, we illustrate with examples how to use PhosphOrtholog to map novel PTM sites from cross-species MS-based phosphoproteomics data.

  18. Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

    PubMed Central

    Gonzales, Patricia A.; Pisitkun, Trairak; Hoffert, Jason D.; Tchapyjnikov, Dmitry; Star, Robert A.; Kleta, Robert; Wang, Nam Sun; Knepper, Mark A.

    2009-01-01

    Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. PMID:19056867

  19. Large-scale proteomics and phosphoproteomics of urinary exosomes.

    PubMed

    Gonzales, Patricia A; Pisitkun, Trairak; Hoffert, Jason D; Tchapyjnikov, Dmitry; Star, Robert A; Kleta, Robert; Wang, Nam Sun; Knepper, Mark A

    2009-02-01

    Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.

  20. Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

    Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis. PMID:24687451

  1. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

    DOE PAGES

    Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

    2016-11-03

    The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

  2. An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

    SciTech Connect

    Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

    2016-11-03

    The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.

  3. Phosphoproteomic analysis of the non-seed vascular plant model Selaginella moellendorffii

    PubMed Central

    2014-01-01

    Background Selaginella (Selaginella moellendorffii) is a lycophyte which diverged from other vascular plants approximately 410 million years ago. As the first reported non-seed vascular plant genome, Selaginella genome data allow comparative analysis of genetic changes that may be associated with land plant evolution. Proteomics investigations on this lycophyte model have not been extensively reported. Phosphorylation represents the most common post-translational modifications and it is a ubiquitous regulatory mechanism controlling the functional expression of proteins inside living organisms. Results In this study, polyethylene glycol fractionation and immobilized metal ion affinity chromatography were employed to isolate phosphopeptides from wild-growing Selaginella. Using liquid chromatography-tandem mass spectrometry analysis, 1593 unique phosphopeptides spanning 1104 non-redundant phosphosites with confirmed localization on 716 phosphoproteins were identified. Analysis of the Selaginella dataset revealed features that are consistent with other plant phosphoproteomes, such as the relative proportions of phosphorylated Ser, Thr, and Tyr residues, the highest occurrence of phosphosites in the C-terminal regions of proteins, and the localization of phosphorylation events outside protein domains. In addition, a total of 97 highly conserved phosphosites in evolutionary conserved proteins were identified, indicating the conservation of phosphorylation-dependent regulatory mechanisms in phylogenetically distinct plant species. On the other hand, close examination of proteins involved in photosynthesis revealed phosphorylation events which may be unique to Selaginella evolution. Furthermore, phosphorylation motif analyses identified Pro-directed, acidic, and basic signatures which are recognized by typical protein kinases in plants. A group of Selaginella-specific phosphoproteins were found to be enriched in the Pro-directed motif class. Conclusions Our work provides

  4. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

    PubMed Central

    Šalovská, Barbora; Fabrik, Ivo; Ďurišová, Kamila; Link, Marek; Vávrová, Jiřina; Řezáčová, Martina; Tichý, Aleš

    2014-01-01

    DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells. PMID:25003641

  5. Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Siminelakis, Paraskeuas; Chairakaki, Aikaterini D; Saez-Rodriguez, Julio; Alexopoulos, Leonidas G

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on

  6. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    PubMed

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. ICT-Based Dynamic Assessment to Reveal Special Education Students' Potential in Mathematics

    ERIC Educational Resources Information Center

    Peltenburg, Marjolijn; van den Heuvel-Panhuizen, Marja; Robitzsch, Alexander

    2010-01-01

    This paper reports on a research project on information and communication technology (ICT)-based dynamic assessment. The project aims to reveal the mathematical potential of students in special education. The focus is on a topic that is generally recognised as rather difficult for weak students: subtraction up to 100 with crossing the ten. The…

  8. A novel dynamics combination model reveals the hidden information of community structure

    NASA Astrophysics Data System (ADS)

    Li, Hui-Jia; Li, Huiying; Jia, Chuanliang

    2015-09-01

    The analysis of the dynamic details of community structure is an important question for scientists from many fields. In this paper, we propose a novel Markov-Potts framework to uncover the optimal community structures and their stabilities across multiple timescales. Specifically, we model the Potts dynamics to detect community structure by a Markov process, which has a clear mathematical explanation. Then the local uniform behavior of spin values revealed by our model is shown that can naturally reveal the stability of hierarchical community structure across multiple timescales. To prove the validity, phase transition of stochastic dynamic system is used to indicate that the stability of community structure we proposed is able to describe the significance of community structure based on eigengap theory. Finally, we test our framework on some example networks and find it does not have resolute limitation problem at all. Results have shown the model we proposed is able to uncover hierarchical structure in different scales effectively and efficiently.

  9. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos.

    PubMed

    Bothma, Jacques P; Garcia, Hernan G; Esposito, Emilia; Schlissel, Gavin; Gregor, Thomas; Levine, Michael

    2014-07-22

    We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4-10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.

  10. Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Wang, Lianshui; Renzi, Emily; Radivojac, Predrag; Tang, Haixu; Arnold, Randy; Zhu, Jian-Kang; Tao, W. Andy

    2013-01-01

    Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants. PMID:23660473

  11. Dynamic changes in network synchrony reveal resting-state functional networks

    NASA Astrophysics Data System (ADS)

    Vuksanović, Vesna; Hövel, Philipp

    2015-02-01

    Experimental functional magnetic resonance imaging studies have shown that spontaneous brain activity, i.e., in the absence of any external input, exhibit complex spatial and temporal patterns of co-activity between segregated brain regions. These so-called large-scale resting-state functional connectivity networks represent dynamically organized neural assemblies interacting with each other in a complex way. It has been suggested that looking at the dynamical properties of complex patterns of brain functional co-activity may reveal neural mechanisms underlying the dynamic changes in functional interactions. Here, we examine how global network dynamics is shaped by different network configurations, derived from realistic brain functional interactions. We focus on two main dynamics measures: synchrony and variations in synchrony. Neural activity and the inferred hemodynamic response of the network nodes are simulated using a system of 90 FitzHugh-Nagumo neural models subject to system noise and time-delayed interactions. These models are embedded into the topology of the complex brain functional interactions, whose architecture is additionally reduced to its main structural pathways. In the simulated functional networks, patterns of correlated regional activity clearly arise from dynamical properties that maximize synchrony and variations in synchrony. Our results on the fast changes of the level of the network synchrony also show how flexible changes in the large-scale network dynamics could be.

  12. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis*

    PubMed Central

    Queiroz, Rayner M. L.; Charneau, Sébastien; Mandacaru, Samuel C.; Schwämmle, Veit; Lima, Beatriz D.; Roepstorff, Peter; Ricart, Carlos A. O.

    2014-01-01

    Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. PMID:25225356

  13. Phosphoproteomic analysis of Her2/neu signaling and inhibition

    PubMed Central

    Bose, Ron; Molina, Henrik; Patterson, A. Scott; Bitok, John K.; Periaswamy, Balamurugan; Bader, Joel S.; Pandey, Akhilesh; Cole, Philip A.

    2006-01-01

    Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20–30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein–protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins. PMID:16785428

  14. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

    Grimes, Mark L.; Lee, Wan-Jui; van der Maaten, Laurens; Shannon, Paul

    2013-01-01

    The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software

  15. Phosphoproteomic Profiling of Selenate-Treated Alzheimer's Disease Model Cells

    PubMed Central

    Wang, Yong; Li, Shuiming; Shen, Liming; Liu, Qiong; Ni, Jiazuan

    2014-01-01

    The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimer's disease (AD). One of the hallmarks of AD is the formation of neurofibrillary tangles (NFTs), which is composed of hyperphosphorylated tau proteins. Sodium selenate has been recently found to reduce tau hyperphosphorylation and NFTs formation, and to improve spatial learning and motor performance in AD mice. In the current study, the phosphoproteomics of N2aSW cells treated with selenate were investigated. To avoid missing low-abundance phosphoproteins, both the total proteins of cells and the phosphor-enriched proteins were extracted and subjected to the two-dimensional gel electrophoresis with Pro-Q diamond staining and then LC-MS/MS analysis. A total of 65 proteins were altered in phosphorylation level, of which 39 were up-regulated and 26 were down-regulated. All identified phosphoproteins were bioinformatically annotated according to their physiochemical features, subcellular location, and biological function. Most of these significantly changed phosphoproteins are involved in crucial neural processes such as protesome activity, oxidative stress, cysteine and methionine metabolism, and energy metabolism. Furthermore, decreases were found in homocysteine, phosphor-tau and amyloid β upon selenate treatment. Our results suggest that selenate may intervene in the pathological process of AD by altering the phosphorylation of some key proteins involved in oxidative stress, energy metabolism and protein degradation, thus play important roles in maintaining redox homeostasis, generating ATP, and clearing misfolded proteins and aggregates. The present paper provides some new clues to the mechanism of selenate in AD prevention. PMID:25485856

  16. Dynamics of methane ebullition from a peat monolith revealed from a dynamic flux chamber system

    NASA Astrophysics Data System (ADS)

    Yu, Zhongjie; Slater, Lee D.; Schäfer, Karina V. R.; Reeve, Andrew S.; Varner, Ruth K.

    2014-09-01

    Methane (CH4) ebullition in northern peatlands is poorly quantified in part due to its high spatiotemporal variability. In this study, a dynamic flux chamber (DFC) system was used to continuously measure CH4 fluxes from a monolith of near-surface Sphagnum peat at the laboratory scale to understand the complex behavior of CH4 ebullition. Coincident transmission ground penetrating radar measurements of gas content were also acquired at three depths within the monolith. A graphical method was developed to separate diffusion, steady ebullition, and episodic ebullition fluxes from the total CH4 flux recorded and to identify the timing and CH4 content of individual ebullition events. The results show that the application of the DFC had minimal disturbance on air-peat CH4 exchange and estimated ebullition fluxes were not sensitive to the uncertainties associated with the graphical model. Steady and episodic ebullition fluxes were estimated to be averagely 36 ± 24% and 38 ± 24% of the total fluxes over the study period, respectively. The coupling between episodic CH4 ebullition and gas content within the three layers supports the existence of a threshold gas content regulating CH4 ebullition. However, the threshold at which active ebullition commenced varied between peat layers with a larger threshold (0.14 m3 m-3) observed in the deeper layers, suggesting that the peat physical structure controls gas bubble dynamics in peat. Temperature variation (23°C to 27°C) was likely only responsible for small episodic ebullition events from the upper peat layer, while large ebullition events from the deeper layers were most likely triggered by drops in atmospheric pressure.

  17. Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons

    PubMed Central

    Siddoway, Benjamin; Hou, Hailong; Yang, Hongtian; Petralia, Ronald; Xia, Houhui

    2013-01-01

    Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically-driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated (ATM) can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that ATM and its sister kinase ATR up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally-regulated by synaptic activity. PMID:24117848

  18. Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation.

    PubMed

    Guo, Mingquan; Huang, Bill X

    2013-02-01

    Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, subcellular translocation, and activation of cellular enzymes. MS-based phosphoproteomics has now allowed the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, which has posed new challenges in functional analysis of each and every phosphorylation site on specific signaling phosphoproteins of interest. In this article, we review recent advances in the functional analysis of targeted phosphorylation carried out by various chemical and biological approaches in combination with the MS-based phosphoproteomics. This review focuses on three types of strategies, including forward functional analysis, defined for the result-driven phosphoproteomics efforts in determining the substrates of a specific protein kinase; reverse functional analysis, defined for tracking the kinase(s) for specific phosphosite(s) derived from the discovery-driven phosphoproteomics efforts; and MS-based analysis on the structure-function relationship of phosphoproteins. It is expected that this review will provide a state-of-the-art overview of functional analysis of site-specific phosphorylation and explore new perspectives and outline future challenges.

  19. Revealing glacier flow and surge dynamics from animated satellite image sequences: examples from the Karakoram

    NASA Astrophysics Data System (ADS)

    Paul, F.

    2015-04-01

    Although animated images are very popular on the Internet, they have so far found only limited use for glaciological applications. With long time-series of satellite images becoming increasingly available and glaciers being well recognized for their rapid changes and variable flow dynamics, animated sequences of multiple satellite images reveal glacier dynamics in a time-lapse mode, making the otherwise slow changes of glacier movement visible and understandable for a wide public. For this study animated image sequences were created from freely available image quick-looks of orthorectified Landsat scenes for four regions in the central Karakoram mountain range. The animations play automatically in a web-browser and might help to demonstrate glacier flow dynamics for educational purposes. The animations revealed highly complex patterns of glacier flow and surge dynamics over a 15-year time period (1998-2013). In contrast to other regions, surging glaciers in the Karakoram are often small (around 10 km2), steep, debris free, and advance for several years at comparably low annual rates (a few hundred m a-1). The advance periods of individual glaciers are generally out of phase, indicating a limited climatic control on their dynamics. On the other hand, nearly all other glaciers in the region are either stable or slightly advancing, indicating balanced or even positive mass budgets over the past few years to decades.

  20. Combined TMS and FMRI reveal dissociable cortical pathways for dynamic and static face perception.

    PubMed

    Pitcher, David; Duchaine, Bradley; Walsh, Vincent

    2014-09-08

    Faces contain structural information, for identifying individuals, as well as changeable information, which can convey emotion and direct attention. Neuroimaging studies reveal brain regions that exhibit preferential responses to invariant [1, 2] or changeable [3-5] facial aspects but the functional connections between these regions are unknown. We addressed this issue by causally disrupting two face-selective regions with thetaburst transcranial magnetic stimulation (TBS) and measuring the effects of this disruption in local and remote face-selective regions with functional magnetic resonance imaging (fMRI). Participants were scanned, over two sessions, while viewing dynamic or static faces and objects. During these sessions, TBS was delivered over the right occipital face area (rOFA) or right posterior superior temporal sulcus (rpSTS). Disruption of the rOFA reduced the neural response to both static and dynamic faces in the downstream face-selective region in the fusiform gyrus. In contrast, the response to dynamic and static faces was doubly dissociated in the rpSTS. Namely, disruption of the rOFA reduced the response to static but not dynamic faces, while disruption of the rpSTS itself reduced the response to dynamic but not static faces. These results suggest that dynamic and static facial aspects are processed via dissociable cortical pathways that begin in early visual cortex, a conclusion inconsistent with current models of face perception [6-9].

  1. Entangled Valence Electron-Hole Dynamics Revealed by Stimulated Attosecond X-ray Raman Scattering

    PubMed Central

    Healion, Daniel; Zhang, Yu; Biggs, Jason D.; Govind, Niranjan

    2012-01-01

    We show that broadband x-ray pulses can create wavepackets of valence electrons and holes localized in the vicinity of a selected atom (nitrogen, oxygen or sulfur in cysteine) by stimulated resonant Raman scattering. The subsequent dynamics reveals highly correlated motions of entangled electrons and hole quasiparticles. This information goes beyond the time-dependent total charge density derived from x-ray diffraction. PMID:23755318

  2. Light-induced nuclear export reveals rapid dynamics of epigenetic modifications

    PubMed Central

    Yumerefendi, Hayretin; Lerner, Andrew Michael; Zimmerman, Seth Parker; Hahn, Klaus; Bear, James E; Strahl, Brian D.; Kuhlman, Brian

    2016-01-01

    We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation. PMID:27089030

  3. Entangled valence electron-hole dynamics revealed by stimulated attosecond x-ray Raman scattering

    SciTech Connect

    Healion, Daniel; Zhang, Yu; Biggs, Jason D.; Govind, Niranjan; Mukamel, Shaul

    2012-09-06

    We show that broadband x-ray pulses can create wavepackets of valence electrons and holes localized in the vicinity of a selected atom (nitrogen, oxygen or sulfur in cysteine) by resonant stimulated Raman scattering. The subsequent dynamics reveals highly correlated motions of entangled electrons and hole quasiparticles. This information goes beyond the time-dependent total charge density derived from x-ray diffraction.

  4. Entangled Valence Electron-Hole Dynamics Revealed by Stimulated Attosecond X-ray Raman Scattering.

    PubMed

    Healion, Daniel; Zhang, Yu; Biggs, Jason D; Govind, Niranjan; Mukamel, Shaul

    2012-08-08

    We show that broadband x-ray pulses can create wavepackets of valence electrons and holes localized in the vicinity of a selected atom (nitrogen, oxygen or sulfur in cysteine) by stimulated resonant Raman scattering. The subsequent dynamics reveals highly correlated motions of entangled electrons and hole quasiparticles. This information goes beyond the time-dependent total charge density derived from x-ray diffraction.

  5. Aerial photographs reveal late-20th-century dynamic ice loss in northwestern Greenland.

    PubMed

    Kjær, Kurt H; Khan, Shfaqat A; Korsgaard, Niels J; Wahr, John; Bamber, Jonathan L; Hurkmans, Ruud; van den Broeke, Michiel; Timm, Lars H; Kjeldsen, Kristian K; Bjørk, Anders A; Larsen, Nicolaj K; Jørgensen, Lars Tyge; Færch-Jensen, Anders; Willerslev, Eske

    2012-08-03

    Global warming is predicted to have a profound impact on the Greenland Ice Sheet and its contribution to global sea-level rise. Recent mass loss in the northwest of Greenland has been substantial. Using aerial photographs, we produced digital elevation models and extended the time record of recent observed marginal dynamic thinning back to the mid-1980s. We reveal two independent dynamic ice loss events on the northwestern Greenland Ice Sheet margin: from 1985 to 1993 and 2005 to 2010, which were separated by limited mass changes. Our results suggest that the ice mass changes in this sector were primarily caused by short-lived dynamic ice loss events rather than changes in the surface mass balance. This finding challenges predictions about the future response of the Greenland Ice Sheet to increasing global temperatures.

  6. More Dynamical Properties Revealed from a 3D Lorenz-like System

    NASA Astrophysics Data System (ADS)

    Wang, Haijun; Li, Xianyi

    After a 3D Lorenz-like system has been revisited, more rich hidden dynamics that was not found previously is clearly revealed. Some more precise mathematical work, such as for the complete distribution and the local stability and bifurcation of its equilibrium points, the existence of singularly degenerate heteroclinic cycles as well as homoclinic and heteroclinic orbits, and the dynamics at infinity, is carried out in this paper. In particular, another possible new mechanism behind the creation of chaotic attractors is presented. Based on this mechanism, some different structure types of chaotic attractors are numerically found in the case of small b > 0. All theoretical results obtained are further illustrated by numerical simulations. What we formulate in this paper is to not only show those dynamical properties hiding in this system, but also (more mainly) present a kind of way and means — both "locally" and "globally" and both "finitely" and "infinitely" — to comprehensively explore a given system.

  7. Multiannual forecasting of seasonal influenza dynamics reveals climatic and evolutionary drivers.

    PubMed

    Axelsen, Jacob Bock; Yaari, Rami; Grenfell, Bryan T; Stone, Lewi

    2014-07-01

    Human influenza occurs annually in most temperate climatic zones of the world, with epidemics peaking in the cold winter months. Considerable debate surrounds the relative role of epidemic dynamics, viral evolution, and climatic drivers in driving year-to-year variability of outbreaks. The ultimate test of understanding is prediction; however, existing influenza models rarely forecast beyond a single year at best. Here, we use a simple epidemiological model to reveal multiannual predictability based on high-quality influenza surveillance data for Israel; the model fit is corroborated by simple metapopulation comparisons within Israel. Successful forecasts are driven by temperature, humidity, antigenic drift, and immunity loss. Essentially, influenza dynamics are a balance between large perturbations following significant antigenic jumps, interspersed with nonlinear epidemic dynamics tuned by climatic forcing.

  8. Dynamic functional network connectivity reveals unique and overlapping profiles of insula subdivisions.

    PubMed

    Nomi, Jason S; Farrant, Kristafor; Damaraju, Eswar; Rachakonda, Srinivas; Calhoun, Vince D; Uddin, Lucina Q

    2016-05-01

    The human insular cortex consists of functionally diverse subdivisions that engage during tasks ranging from interoception to cognitive control. The multiplicity of functions subserved by insular subdivisions calls for a nuanced investigation of their functional connectivity profiles. Four insula subdivisions (dorsal anterior, dAI; ventral, VI; posterior, PI; middle, MI) derived using a data-driven approach were subjected to static- and dynamic functional network connectivity (s-FNC and d-FNC) analyses. Static-FNC analyses replicated previous work demonstrating a cognition-emotion-interoception division of the insula, where the dAI is functionally connected to frontal areas, the VI to limbic areas, and the PI and MI to sensorimotor areas. Dynamic-FNC analyses consisted of k-means clustering of sliding windows to identify variable insula connectivity states. The d-FNC analysis revealed that the most frequently occurring dynamic state mirrored the cognition-emotion-interoception division observed from the s-FNC analysis, with less frequently occurring states showing overlapping and unique subdivision connectivity profiles. In two of the states, all subdivisions exhibited largely overlapping profiles, consisting of subcortical, sensory, motor, and frontal connections. Two other states showed the dAI exhibited a unique connectivity profile compared with other insula subdivisions. Additionally, the dAI exhibited the most variable functional connections across the s-FNC and d-FNC analyses, and was the only subdivision to exhibit dynamic functional connections with regions of the default mode network. These results highlight how a d-FNC approach can capture functional dynamics masked by s-FNC approaches, and reveal dynamic functional connections enabling the functional flexibility of the insula across time. Hum Brain Mapp 37:1770-1787, 2016. © 2016 Wiley Periodicals, Inc.

  9. Effects of pressure on the dynamics of a hyperthermophilic protein revealed by quasielastic neutron scattering

    NASA Astrophysics Data System (ADS)

    Shrestha, U. R.; Bhowmik, D.; Copley, J. R. D.; Tyagi, M.; Leao, J. B.; Chu, X.-Q.

    Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is nearly 100 MPa. Here we study the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range using quasielastic neutron scattering (QENS) technique. Two spectrometers were used to investigate the β-relaxation dynamics of proteins in time ranges from 2 to 25 ps, and from 100 ps to 2 ns. Our results reveal that, under the pressure of 100 MPa, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), opposite to what we observed previously under the ambient pressure. These contradictory observations imply that high pressure affects the dynamical properties of proteins by distorting their energy landscapes. Accordingly, we derived a general schematic denaturation phase diagram that can be used as a general picture to understand the effects of pressure on protein dynamics and activities Wayne State Univ Startup Fund.

  10. Structure and dynamics of protein waters revealed by radiolysis and mass spectrometry

    PubMed Central

    Gupta, Sayan; D’Mello, Rhijuta; Chance, Mark R.

    2012-01-01

    Water is critical for the structure, stability, and functions of macromolecules. Diffraction and NMR studies have revealed structure and dynamics of bound waters at atomic resolution. However, localizing the sites and measuring the dynamics of bound waters, particularly on timescales relevant to catalysis and macromolecular assembly, is quite challenging. Here we demonstrate two techniques: first, temperature-dependent radiolytic hydroxyl radical labeling with a mass spectrometry (MS)-based readout to identify sites of bulk and bound water interactions with surface and internal residue side chains, and second, H218O radiolytic exchange coupled MS to measure the millisecond dynamics of bound water interactions with various internal residue side chains. Through an application of the methods to cytochrome c and ubiquitin, we identify sites of water binding and measure the millisecond dynamics of bound waters in protein crevices. As these MS-based techniques are very sensitive and not protein size limited, they promise to provide unique insights into protein–water interactions and water dynamics for both small and large proteins and their complexes. PMID:22927377

  11. Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

    PubMed

    Salih, Erdjan

    2005-01-01

    spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

  12. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane

    SciTech Connect

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed phosphorylation

  13. Nonlinear elastic behavior of rocks revealed by dynamic acousto-elastic testing

    NASA Astrophysics Data System (ADS)

    Shokouhi, Parisa; Riviere, Jacques; Guyer, Robert; Johnson, Paul

    2017-04-01

    Nonlinear elastic behavior of rocks is studied at the laboratory scale with the goal of illuminating observations at the Earth scale, for instance during strong ground motion and earthquake slip processes. A technique called Dynamic Acousto-Elastic Testing (DAET) is used to extract the nonlinear elastic response of disparate rocks (sandstone, granite and soapstone). DAET is the dynamic analogous to standard (quasi-static) acousto-elastic testing. It consists in measuring speed of sound with high-frequency low amplitude pulses (MHz range) across the sample while it is dynamically loaded with a low frequency, large amplitude resonance (kHz range). This particular configuration provides the instantaneous elastic response over a full dynamic cycle and reveals unprecedented details: instantaneous softening, tension/compression asymmetry as well as hysteretic behaviors. The strain-induced modulation of ultrasonic pulse velocities ('fast dynamics') is analyzed to extract nonlinearity parameters. A projection method is used to extract the harmonic content and a careful comparison of the fast dynamics response is made. In order to characterize the rate of elastic recovery ('slow dynamics'), we continue to monitor the ultrasonic wave velocity for about 30 minutes after the low-frequency resonance is turned off. In addition, the frequency, pressure and humidity dependences of the nonlinear parameters are reported for a subset of samples. We find that the nonlinear components can be clustered into two categories, which suggests that two main mechanisms are at play. The first one, related to the second harmonic, is likely related to the opening/closing of microstructural features such as cracks and grain/grain contacts. In contrast, the second mechanism is related to all other nonlinear parameters (transient softening, hysteresis area and higher order harmonics) and may arise from shearing mechanisms at grain interfaces.

  14. Dynamic processes in biological membrane mimics revealed by quasielastic neutron scattering.

    PubMed

    Lautner, Lisa; Pluhackova, Kristyna; Barth, Nicolai K H; Seydel, Tilo; Lohstroh, Wiebke; Böckmann, Rainer A; Unruh, Tobias

    2017-08-01

    Neutron scattering is a powerful tool to study relaxation processes in biological membrane mimics in space and time. Combining different inelastic and quasielastic neutron scattering techniques, a large dynamic range can be covered: from atomic to mesoscopic lengths and from femto- to some hundreds of nanoseconds in time. This allows studies on e.g. the diffusion of lipids, the membrane undulation motions, the dispersion of sound waves in membranes as well as the mutual interactions of membrane constituents such as lipids, proteins, and additives. In particular, neutron scattering provides a quite direct experimental approach to the inter-atomic and inter-molecular potentials on length and time scales which are perfectly accessible by molecular dynamics (MD) simulations. Neutron scattering experiments may thus substantially support the further refinement of biomolecular force fields for MD simulations by supplying structural and dynamical information with high spatial and temporal resolution. In turn, MD simulations support the interpretation of neutron scattering data. The combination of both, neutron scattering experiments and MD simulations, yields an unprecedented insight into the molecular interactions governing the structure and dynamics of biological membranes. This review provides an overview of the molecular dynamics in biological membrane mimics as revealed by neutron scattering. It focuses on the latest findings such as the fundamental molecular mechanism of lateral lipid diffusion as well as the influence of additives and proteins on the short-time dynamics of lipids. Special emphasis is placed on the comparison of recent neutron scattering and MD simulation data with respect to molecular membrane dynamics on the pico- to nanosecond time scale. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Visualization of microtubule growth in living platelets reveals a dynamic marginal band with multiple microtubules

    PubMed Central

    Patel-Hett, Sunita; Richardson, Jennifer L.; Schulze, Harald; Drabek, Ksenija; Isaac, Natasha A.; Hoffmeister, Karin; Shivdasani, Ramesh A.; Bulinski, J. Chloë; Galjart, Niels; Hartwig, John H.

    2008-01-01

    The marginal band of microtubules maintains the discoid shape of resting blood platelets. Although studies of platelet microtubule coil structure conclude that it is composed of a single microtubule, no investigations of its dynamics exist. In contrast to previous studies, permeabilized platelets incubated with GTP-rhodamine-tubulin revealed tubulin incorporation at 7.9 (± 1.9) points throughout the coil, and anti-EB1 antibodies stained 8.7 (± 2.0) sites, indicative of multiple free microtubules. To pursue this result, we expressed the microtubule plus-end marker EB3-GFP in megakaryocytes and examined its behavior in living platelets released from these cells. Time-lapse microscopy of EB3-GFP in resting platelets revealed multiple assembly sites within the coil and a bidirectional pattern of assembly. Consistent with these findings, tyrosinated tubulin, a marker of newly assembled microtubules, localized to resting platelet microtubule coils. These results suggest that the resting platelet marginal band contains multiple highly dynamic microtubules of mixed polarity. Analysis of microtubule coil diameters in newly formed resting platelets indicates that microtubule coil shrinkage occurs with aging. In addition, activated EB3-GFP–expressing platelets exhibited a dramatic increase in polymerizing microtubules, which travel outward and into filopodia. Thus, the dynamic microtubules associated with the marginal band likely function during both resting and activated platelet states. PMID:18230754

  16. Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages.

    PubMed

    Dietrich, Jens-Erik; Panavaite, Laura; Gunther, Stefan; Wennekamp, Sebastian; Groner, Anna C; Pigge, Anton; Salvenmoser, Stefanie; Trono, Didier; Hufnagel, Lars; Hiiragi, Takashi

    2015-08-01

    Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability.

  17. Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

    PubMed

    Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F; Puchta, Holger; Houben, Andreas

    2017-08-01

    Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  18. Single-molecule chemical reaction reveals molecular reaction kinetics and dynamics.

    PubMed

    Zhang, Yuwei; Song, Ping; Fu, Qiang; Ruan, Mingbo; Xu, Weilin

    2014-06-25

    Understanding the microscopic elementary process of chemical reactions, especially in condensed phase, is highly desirable for improvement of efficiencies in industrial chemical processes. Here we show an approach to gaining new insights into elementary reactions in condensed phase by combining quantum chemical calculations with a single-molecule analysis. Elementary chemical reactions in liquid-phase, revealed from quantum chemical calculations, are studied by tracking the fluorescence of single dye molecules undergoing a reversible redox process. Statistical analyses of single-molecule trajectories reveal molecular reaction kinetics and dynamics of elementary reactions. The reactivity dynamic fluctuations of single molecules are evidenced and probably arise from either or both of the low-frequency approach of the molecule to the internal surface of the SiO2 nanosphere or the molecule diffusion-induced memory effect. This new approach could be applied to other chemical reactions in liquid phase to gain more insight into their molecular reaction kinetics and the dynamics of elementary steps.

  19. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    PubMed

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

  20. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

    PubMed Central

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  1. Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

    PubMed Central

    2013-01-01

    The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein

  2. Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

    PubMed Central

    Fusco, Ludovico; Lefort, Riwal; Smith, Kevin; Benmansour, Fethallah; Gonzalez, German; Barillari, Caterina; Rinn, Bernd; Fleuret, Francois; Fua, Pascal

    2016-01-01

    Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth. PMID:26728857

  3. Inverting observations of GPS refractivies to reveal dynamical structures for climate model testing

    NASA Astrophysics Data System (ADS)

    Dykema, J. A.; Leroy, S. S.; Farrell, B. F.; Anderson, J. G.

    2004-12-01

    Refractivities derived from measurements of GPS radio occultation, with an optimal choice of orbits, can provide a globally homogeneous record of the state of the climate. These refractivities contribute information in both the troposphere and stratosphere, sensitive to temperature, water vapor, and pressure in all weather conditions. This submission describes a method to extract information directly from these space observations to diagnose climate model dynamics. Although traditionally remotely sensed variables (such as refractivities) have been inverted to produce profiles of more familiar atmospheric state variables such as temperature, pressure, or water vapor, the refractivities themselves provide an ideal state vector for analysis by linear inverse modeling (LIM). The success of LIM for ENSO and seasonal climate forecasting (Penland and Magorian 1993; Winkler et al. 2001) reveals that inverting selected observations for dynamics is a powerful methodology compared with approximating dynamics of complex processes from first principles. Development of this approach based on observation state space reconstruction is motivated in part by the realization that identifying model error and improving model parameterizations is a very difficult task to accomplish by appeal to physical argument and first principle reasoning alone, as the variety of cloud parameterizations testifies. Continued progress in model refinement requires developing methods to systematize parameterization improvement. A benchmark for model improvement, therefore, is that the model reproduce the LIM dynamics in appropriate variables.

  4. Dynamic Localization of Electronic Excitation in Photosynthetic Complexes Revealed with Chiral Two-Dimensional Spectroscopy

    PubMed Central

    Fidler, Andrew F.; Singh, Ved P.; Long, Phillip D.; Dahlberg, Peter D.; Engel, Gregory S.

    2014-01-01

    Time-resolved ultrafast optical probes of chiral dynamics provide a new window allowing us to explore how interactions with such structured environments drive electronic dynamics. Incorporating optical activity into time-resolved spectroscopies has proven challenging due to the small signal and large achiral background. Here, we demonstrate that two-dimensional electronic spectroscopy can be adapted to detect chiral signals and that these signals reveal how excitations delocalize and contract following excitation. We dynamically probe the evolution of chiral electronic structure in the light harvesting complex 2 of purple bacteria following photoexcitation by creating a chiral two-dimensional mapping. The dynamics of the chiral two-dimensional signal directly reports on changes in the degree of delocalization of the excitonic state following photoexcitation. The mechanism of energy transfer in this system may enhance transfer probability due to the coherent coupling among chromophores while suppressing fluorescence that arises from populating delocalized states. This generally applicable spectroscopy will provide an incisive tool to probe ultrafast transient molecular fluctuations that are obscured in non-chiral experiments. PMID:24504144

  5. Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange

    NASA Astrophysics Data System (ADS)

    Lou, Xianwen; Lafleur, René P. M.; Leenders, Christianus M. A.; Schoenmakers, Sandra M. C.; Matsumoto, Nicholas M.; Baker, Matthew B.; van Dongen, Joost L. J.; Palmans, Anja R. A.; Meijer, E. W.

    2017-05-01

    Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.

  6. Ligation-Dependent Picosecond Dynamics in Human Hemoglobin As Revealed by Quasielastic Neutron Scattering.

    PubMed

    Fujiwara, Satoru; Chatake, Toshiyuki; Matsuo, Tatsuhito; Kono, Fumiaki; Tominaga, Taiki; Shibata, Kaoru; Sato-Tomita, Ayana; Shibayama, Naoya

    2017-08-31

    Hemoglobin, the vital O2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

  7. Cape buffalo mitogenomics reveals a Holocene shift in the African human-megafauna dynamics.

    PubMed

    Heller, Rasmus; Brüniche-Olsen, Anna; Siegismund, Hans R

    2012-08-01

    Africa is unique among the continents in having maintained an extraordinarily diverse and prolific megafauna spanning the Pleistocene-Holocene epochs. Little is known about the historical dynamics of this community and even less about the reasons for its unique persistence to modern times. We sequenced complete mitochondrial genomes from 43 Cape buffalo (Syncerus caffer caffer) to infer the demographic history of this large mammal. A combination of Bayesian skyline plots, simulations and Approximate Bayesian Computation (ABC) were used to distinguish population size dynamics from the confounding effect of population structure and identify the most probable demographic scenario. Our analyses revealed a late Pleistocene expansion phase concurrent with the human expansion between 80 000 and 10 000 years ago, refuting an adverse ecological effect of Palaeolithic humans on this quarry species, but also showed that the buffalo subsequently declined during the Holocene. The distinct two-phased dynamic inferred here suggests that a major ecological transition occurred in the Holocene. The timing of this transition coincides with the onset of drier conditions throughout tropical Africa following the Holocene Optimum (∼9000-5000 years ago), but also with the explosive growth in human population size associated with the transition from the Palaeolithic to the Neolithic cultural stage. We evaluate each of these possible causal factors and their potential impact on the African megafauna, providing the first systematic assessment of megafauna dynamics on the only continent where large mammals remain abundant.

  8. Dynamic diversity of synthetic supramolecular polymers in water as revealed by hydrogen/deuterium exchange.

    PubMed

    Lou, Xianwen; Lafleur, René P M; Leenders, Christianus M A; Schoenmakers, Sandra M C; Matsumoto, Nicholas M; Baker, Matthew B; van Dongen, Joost L J; Palmans, Anja R A; Meijer, E W

    2017-05-15

    Numerous self-assembling molecules have been synthesized aiming at mimicking both the structural and dynamic properties found in living systems. Here we show the application of hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to unravel the nanoscale organization and the structural dynamics of synthetic supramolecular polymers in water. We select benzene-1,3,5-tricarboxamide (BTA) derivatives that self-assemble in H2O to illustrate the strength of this technique for supramolecular polymers. The BTA structure has six exchangeable hydrogen atoms and we follow their exchange as a function of time after diluting the H2O solution with a 100-fold excess of D2O. The kinetic H/D exchange profiles reveal that these supramolecular polymers in water are dynamically diverse; a notion that has previously not been observed using other techniques. In addition, we report that small changes in the molecular structure can be used to control the dynamics of synthetic supramolecular polymers in water.

  9. Capturing Arabidopsis root architecture dynamics with ROOT-FIT reveals diversity in responses to salinity.

    PubMed

    Julkowska, Magdalena M; Hoefsloot, Huub C J; Mol, Selena; Feron, Richard; de Boer, Gert-Jan; Haring, Michel A; Testerink, Christa

    2014-11-01

    The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na(+)/K(+) ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked.

  10. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

    PubMed Central

    Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.

    2013-01-01

    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation. PMID:23349924

  11. Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

    PubMed Central

    He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J.; Lu, H. Peter

    2015-01-01

    Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS. PMID:26311846

  12. Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

    PubMed Central

    Moreira-Leite, Flavia; Varga, Vladimir; Gull, Keith

    2016-01-01

    The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics. PMID:27519801

  13. A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants.

    PubMed

    Larrieu, Antoine; Champion, Antony; Legrand, Jonathan; Lavenus, Julien; Mast, David; Brunoud, Géraldine; Oh, Jaesung; Guyomarc'h, Soazig; Pizot, Maxime; Farmer, Edward E; Turnbull, Colin; Vernoux, Teva; Bennett, Malcolm J; Laplaze, Laurent

    2015-01-16

    Activated forms of jasmonic acid (JA) are central signals coordinating plant responses to stresses, yet tools to analyse their spatial and temporal distribution are lacking. Here we describe a JA perception biosensor termed Jas9-VENUS that allows the quantification of dynamic changes in JA distribution in response to stress with high spatiotemporal sensitivity. We show that Jas9-VENUS abundance is dependent on bioactive JA isoforms, the COI1 co-receptor, a functional Jas motif and proteasome activity. We demonstrate the utility of Jas9-VENUS to analyse responses to JA in planta at a cellular scale, both quantitatively and dynamically. This included using Jas9-VENUS to determine the cotyledon-to-root JA signal velocities on wounding, revealing two distinct phases of JA activity in the root. Our results demonstrate the value of developing quantitative sensors such as Jas9-VENUS to provide high-resolution spatiotemporal data about hormone distribution in response to plant abiotic and biotic stresses.

  14. Revealing glacier flow and surge dynamics from animated satellite image sequences: examples from the Karakoram

    NASA Astrophysics Data System (ADS)

    Paul, F.

    2015-11-01

    Although animated images are very popular on the internet, they have so far found only limited use for glaciological applications. With long time series of satellite images becoming increasingly available and glaciers being well recognized for their rapid changes and variable flow dynamics, animated sequences of multiple satellite images reveal glacier dynamics in a time-lapse mode, making the otherwise slow changes of glacier movement visible and understandable to the wider public. For this study, animated image sequences were created for four regions in the central Karakoram mountain range over a 25-year time period (1990-2015) from freely available image quick-looks of orthorectified Landsat scenes. The animations play automatically in a web browser and reveal highly complex patterns of glacier flow and surge dynamics that are difficult to obtain by other methods. In contrast to other regions, surging glaciers in the Karakoram are often small (10 km2 or less), steep, debris-free, and advance for several years to decades at relatively low annual rates (about 100 m a-1). These characteristics overlap with those of non-surge-type glaciers, making a clear identification difficult. However, as in other regions, the surging glaciers in the central Karakoram also show sudden increases of flow velocity and mass waves travelling down glacier. The surges of individual glaciers are generally out of phase, indicating a limited climatic control on their dynamics. On the other hand, nearly all other glaciers in the region are either stable or slightly advancing, indicating balanced or even positive mass budgets over the past few decades.

  15. Identification of novel signaling components in N,N’-Dinitrosopiperazine-mediated metastasis of nasopharyngeal Carcinoma by quantitative phosphoproteomics

    PubMed Central

    2014-01-01

    Background Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic cancer. N,N’-dinitrosopiperazine (DNP), a carcinogen with specificity for nasopharyngeal epithelium, facilitates NPC metastasis. However, the underlying mechanism is not known. Methods Quantitative phosphoproteomics, using stable isotope labeling of amino acids in cell cultures, was employed to identify phosphoproteins associated with NPC metastasis mediated by DNP. NPC cell line 6-10B, which is relatively less metastatic, was used to investigate DNP-mediated metastasis. Boyden chamber invasion assay was used to measure DNP-induced motility and invasion, and nude mice were used to verify DNP-mediated metastasis in vivo. Several different phosphoproteins detected by proteomics analysis were verified by immunoblotting. DNP-mediated metastasis facilitated by lysine-rich CEACAM1 co-isolated protein (LYRIC) phosphorylation at serine 568 was confirmed using mutations targeting the phosphorylation site of LYRIC. DNP-mediated metastasis through LYRIC phosphorylation was confirmed in the NPC cell line CNE1. DNP-mediated LYRIC phosphorylation at serine 568 was also verified in metastatic tumors of BABL/c nude mice. Results Boyden chamber invasion assay indicated that DNP mediated cell motility and invasion of NPC cell 6-10B in vitro, and experiments with nude mice indicated that DNP increased 6-10B metastasis in vivo. In the phosphoproteomics analysis, we detected 216 phosphorylation sites on 130 proteins; among these, 48 phosphorylation sites on 30 unique phosphopeptides were modulated by DNP by at least 1.5-fold. DNP mediated the expression of phosphorylated GTPase, ferritin, LYRIC, and RNA polymerase, and it decreased the expression of phosphorylated torsin-1A protein 1. Furthermore, DNP induced LYRIC phosphorylation at serine 568 to facilitate cell motility and invasion, whereas DNP-mediated motility and invasion was decreased when serine 568 in LYRIC was mutated. In another NPC cell line

  16. Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation.

    PubMed

    Maier, Ezekiel J; Haynes, Brian C; Gish, Stacey R; Wang, Zhuo A; Skowyra, Michael L; Marulli, Alyssa L; Doering, Tamara L; Brent, Michael R

    2015-05-01

    Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus-its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and "foreman" TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes.

  17. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation.

    PubMed

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-05-26

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

  18. Dataset of the Botrytis cinerea phosphoproteome induced by different plant-based elicitors.

    PubMed

    Liñeiro, Eva; Chiva, Cristina; Cantoral, Jesús M; Sabido, Eduard; Fernández-Acero, Francisco Javier

    2016-06-01

    Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled "Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors" (Liñeiro et al., 2016) [1].

  19. Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma

    PubMed Central

    Parker, Robert; Vella, Laura J.; Xavier, Dylan; Amirkhani, Ardeshir; Parker, Jimmy; Cebon, Jonathan; Molloy, Mark P.

    2015-01-01

    The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype. PMID:26029660

  20. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    PubMed

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  1. EEG microstate sequences in healthy humans at rest reveal scale-free dynamics

    PubMed Central

    Van De Ville, Dimitri; Britz, Juliane; Michel, Christoph M.

    2010-01-01

    Recent findings identified electroencephalography (EEG) microstates as the electrophysiological correlates of fMRI resting-state networks. Microstates are defined as short periods (100 ms) during which the EEG scalp topography remains quasi-stable; that is, the global topography is fixed but strength might vary and polarity invert. Microstates represent the subsecond coherent activation within global functional brain networks. Surprisingly, these rapidly changing EEG microstates correlate significantly with activity in fMRI resting-state networks after convolution with the hemodynamic response function that constitutes a strong temporal smoothing filter. We postulate here that microstate sequences should reveal scale-free, self-similar dynamics to explain this remarkable effect and thus that microstate time series show dependencies over long time ranges. To that aim, we deploy wavelet-based fractal analysis that allows determining scale-free behavior. We find strong statistical evidence that microstate sequences are scale free over six dyadic scales covering the 256-ms to 16-s range. The degree of long-range dependency is maintained when shuffling the local microstate labels but becomes indistinguishable from white noise when equalizing microstate durations, which indicates that temporal dynamics are their key characteristic. These results advance the understanding of temporal dynamics of brain-scale neuronal network models such as the global workspace model. Whereas microstates can be considered the “atoms of thoughts,” the shortest constituting elements of cognition, they carry a dynamic signature that is reminiscent at characteristic timescales up to multiple seconds. The scale-free dynamics of the microstates might be the basis for the rapid reorganization and adaptation of the functional networks of the brain. PMID:20921381

  2. StageTip-based HAMMOC, an efficient and inexpensive phosphopeptide enrichment method for plant shotgun phosphoproteomics.

    PubMed

    Nakagami, Hirofumi

    2014-01-01

    Phosphopeptide enrichment is the most critical step for successful LC-MS/MS-based shotgun phosphoproteomics. Recent technological improvements have made selective phosphopeptide enrichment from non-fractionated whole cell lysate digests with a single-step procedure possible. Here, a handy protocol is described for phosphopeptide enrichment from plant materials using hydroxy acid-modified metal oxide chromatography (HAMMMOC) with a stop-and-go-extraction tip (StageTip).

  3. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    DOE PAGES

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; ...

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here,more » we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.« less

  4. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    SciTech Connect

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; van der Meulen, Annelot I.; Borden, Sean V.; van Steensel, Bas; Bindra, Ranjit S.; LaRocque, Jeannine R.; Karpen, Gary H.

    2016-07-15

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

  5. Structural signatures of DRD4 mutants revealed using molecular dynamics simulations: Implications for drug targeting.

    PubMed

    Jatana, Nidhi; Thukral, Lipi; Latha, N

    2016-01-01

    Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.

  6. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

    PubMed Central

    Janssen, Aniek; Breuer, Gregory A.; Brinkman, Eva K.; van der Meulen, Annelot I.; Borden, Sean V.; van Steensel, Bas; Bindra, Ranjit S.; LaRocque, Jeannine R.; Karpen, Gary H.

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio–temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains. PMID:27474442

  7. Dynamic metabolome profiling reveals significant metabolic changes during grain development of bread wheat (Triticum aestivum L.).

    PubMed

    Zhen, Shoumin; Dong, Kun; Deng, Xiong; Zhou, Jiaxing; Xu, Xuexin; Han, Caixia; Zhang, Wenying; Xu, Yanhao; Wang, Zhimin; Yan, Yueming

    2016-08-01

    Metabolites in wheat grains greatly influence nutritional values. Wheat provides proteins, minerals, B-group vitamins and dietary fiber to humans. These metabolites are important to human health. However, the metabolome of the grain during the development of bread wheat has not been studied so far. In this work the first dynamic metabolome of the developing grain of the elite Chinese bread wheat cultivar Zhongmai 175 was analyzed, using non-targeted gas chromatography/mass spectrometry (GC/MS) for metabolite profiling. In total, 74 metabolites were identified over the grain developmental stages. Metabolite-metabolite correlation analysis revealed that the metabolism of amino acids, carbohydrates, organic acids, amines and lipids was interrelated. An integrated metabolic map revealed a distinct regulatory profile. The results provide information that can be used by metabolic engineers and molecular breeders to improve wheat grain quality. The present metabolome approach identified dynamic changes in metabolite levels, and correlations among such levels, in developing seeds. The comprehensive metabolic map may be useful when breeding programs seek to improve grain quality. The work highlights the utility of GC/MS-based metabolomics, in conjunction with univariate and multivariate data analysis, when it is sought to understand metabolic changes in developing seeds. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  8. Ocean Processes Revealing by Seasonal Dynamics of Surface Chlorophyll Concentration (by Satellite Data)

    NASA Astrophysics Data System (ADS)

    Shevyrnogov, Anatoly; Vysotskaya, Galina

    Continuous monitoring of phytopigment concentrations in the ocean by space-borne methods makes possible to estimate ecological condition of biocenoses in critical areas. Unlike land vege-tation, hydrological processes largely determine phytoplankton dynamics, which may be either recurrent or random. The types of chlorophyll concentration dynamics can manifest as zones quasistationary by seasonal chlorophyll dynamics, perennial variations of phytopigment con-centrations, anomalous variations, etc., that makes possible revealing of hydrological structure of the ocean. While large-scale and frequently occurring phenomena have been much studied, the seldom-occurring changes of small size may be of interest for analysis of long-term processes and rare natural variations. Along with this, the ability to reflect consequences of anthropoge-nous impact or natural ecological disasters on the ocean biota makes the anomalous variations ecologically essential. Civilization aspiring for steady development and preservation of the bio-sphere, must have the knowledge of spatial distribution, seasonal dynamics and anomalies of the primary production process on the planet. In the papers of the authors (Shevyrnogov A.P., Vysotskaya G.S., Gitelzon J.I. Quasistationary areas of chlorophyll concentration in the world ocean as observed satellite data. Adv. Space Res. Vol. 18, No. 7, pp. 129-132, 1996) existence of zones, which are quasi-stationary with similar seasonal dynamics of chlorophyll concentration at surface layer of ocean, was shown. Results were obtained on the base of pro-cessing of time series of satellite images SeaWiFS. It was shown that fronts and frontal zones coincide with dividing lines between quasi-stationary areas, especially in areas of large oceanic streams. Biota of surface oceanic layer is more stable in comparison with quickly changing sur-face temperature. It gives a possibility to circumvent influence of high-frequency component (for example, a diurnal cycle

  9. Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana.

    PubMed

    Krahmer, Johanna; Hindle, Matthew M; Martin, Sarah F; Le Bihan, Thierry; Millar, Andrew J

    2015-01-01

    Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract. © 2015 Elsevier Inc. All rights reserved.

  10. Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

  11. Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. PMID:23316220

  12. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    PubMed

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  13. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

    PubMed

    Tan, Haiyan; Wu, Zhiping; Wang, Hong; Bai, Bing; Li, Yuxin; Wang, Xusheng; Zhai, Bo; Beach, Thomas G; Peng, Junmin

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).

  14. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  15. Using State Space Methods to Reveal Dynamical Associations Between Cortisol and Depression.

    PubMed

    Toonen, Roelof B; Wardenaar, Klaas J; van Ockenburg, Sonja L; Bos, Elisabeth H; de Jonge, Peter

    2016-01-01

    Despite extensive research, the link between etiological factors and depression remains poorly understood. This may in part be due to a focus on strictly linear definitions of causality, derived at the group level. However, etiological relations in depression are likely to be dynamical, nonlinear and potentially unquantifiable with traditional statistics. Therefore the aim of this study was to evaluate the use of the convergent cross-mapping (CCM) method in investigating possible nonlinear relationships between supposed etiological factors and depressive symptomatology. Time series data from six healthy individuals were used to model the relationship between 24-h urinary free cortisol and negative affect using CCM and dewdrop embeddings. CCM is a nonlinear measure of causality, based on state space reconstruction with lagged coordinate embeddings. The results showed that nonlinear dynamical relationships between cortisol and negative affect may be present within participants, as demonstrated by a positive cross-map convergence from negative affect to cortisol. However, analyses also showed that noise and influential points had considerable impact on the results. Convergent crossmapping can be used to reveal possible nonlinear dynamical relationships between etiological factors and psychopathology that may remain undetected with traditional linear causality measures.

  16. Complex Contact-Based Dynamics of Microsphere Monolayers Revealed by Resonant Attenuation of Surface Acoustic Waves

    NASA Astrophysics Data System (ADS)

    Hiraiwa, M.; Abi Ghanem, M.; Wallen, S. P.; Khanolkar, A.; Maznev, A. A.; Boechler, N.

    2016-05-01

    Contact-based vibrations play an essential role in the dynamics of granular materials. Significant insights into vibrational granular dynamics have previously been obtained with reduced-dimensional systems containing macroscale particles. We study contact-based vibrations of a two-dimensional monolayer of micron-sized spheres on a solid substrate that forms a microscale granular crystal. Measurements of the resonant attenuation of laser-generated surface acoustic waves reveal three collective vibrational modes that involve displacements and rotations of the microspheres, as well as interparticle and particle-substrate interactions. To identify the modes, we tune the interparticle stiffness, which shifts the frequency of the horizontal-rotational resonances while leaving the vertical resonance unaffected. From the measured contact resonance frequencies we determine both particle-substrate and interparticle contact stiffnesses and find that the former is an order of magnitude larger than the latter. This study paves the way for investigating complex contact-based dynamics of microscale granular crystals and yields a new approach to studying micro- to nanoscale contact mechanics in multiparticle networks.

  17. Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

    NASA Astrophysics Data System (ADS)

    Pageon, Sophie V.; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G.; Davis, Daniel M.

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.

  18. Waiting time distribution revealing the internal spin dynamics in a double quantum dot

    NASA Astrophysics Data System (ADS)

    Ptaszyński, Krzysztof

    2017-07-01

    Waiting time distribution and the zero-frequency full counting statistics of unidirectional electron transport through a double quantum dot molecule attached to spin-polarized leads are analyzed using the quantum master equation. The waiting time distribution exhibits a nontrivial dependence on the value of the exchange coupling between the dots and the gradient of the applied magnetic field, which reveals the oscillations between the spin states of the molecule. The zero-frequency full counting statistics, on the other hand, is independent of the aforementioned quantities, thus giving no insight into the internal dynamics. The fact that the waiting time distribution and the zero-frequency full counting statistics give a nonequivalent information is associated with two factors. Firstly, it can be explained by the sensitivity to different timescales of the dynamics of the system. Secondly, it is associated with the presence of the correlation between subsequent waiting times, which makes the renewal theory, relating the full counting statistics and the waiting time distribution, no longer applicable. The study highlights the particular usefulness of the waiting time distribution for the analysis of the internal dynamics of mesoscopic systems.

  19. Dynamic Changes in Amygdala Psychophysiological Connectivity Reveal Distinct Neural Networks for Facial Expressions of Basic Emotions

    PubMed Central

    Diano, Matteo; Tamietto, Marco; Celeghin, Alessia; Weiskrantz, Lawrence; Tatu, Mona-Karina; Bagnis, Arianna; Duca, Sergio; Geminiani, Giuliano; Cauda, Franco; Costa, Tommaso

    2017-01-01

    The quest to characterize the neural signature distinctive of different basic emotions has recently come under renewed scrutiny. Here we investigated whether facial expressions of different basic emotions modulate the functional connectivity of the amygdala with the rest of the brain. To this end, we presented seventeen healthy participants (8 females) with facial expressions of anger, disgust, fear, happiness, sadness and emotional neutrality and analyzed amygdala’s psychophysiological interaction (PPI). In fact, PPI can reveal how inter-regional amygdala communications change dynamically depending on perception of various emotional expressions to recruit different brain networks, compared to the functional interactions it entertains during perception of neutral expressions. We found that for each emotion the amygdala recruited a distinctive and spatially distributed set of structures to interact with. These changes in amygdala connectional patters characterize the dynamic signature prototypical of individual emotion processing, and seemingly represent a neural mechanism that serves to implement the distinctive influence that each emotion exerts on perceptual, cognitive, and motor responses. Besides these differences, all emotions enhanced amygdala functional integration with premotor cortices compared to neutral faces. The present findings thus concur to reconceptualise the structure-function relation between brain-emotion from the traditional one-to-one mapping toward a network-based and dynamic perspective. PMID:28345642

  20. Dynamic Changes in Amygdala Psychophysiological Connectivity Reveal Distinct Neural Networks for Facial Expressions of Basic Emotions.

    PubMed

    Diano, Matteo; Tamietto, Marco; Celeghin, Alessia; Weiskrantz, Lawrence; Tatu, Mona-Karina; Bagnis, Arianna; Duca, Sergio; Geminiani, Giuliano; Cauda, Franco; Costa, Tommaso

    2017-03-27

    The quest to characterize the neural signature distinctive of different basic emotions has recently come under renewed scrutiny. Here we investigated whether facial expressions of different basic emotions modulate the functional connectivity of the amygdala with the rest of the brain. To this end, we presented seventeen healthy participants (8 females) with facial expressions of anger, disgust, fear, happiness, sadness and emotional neutrality and analyzed amygdala's psychophysiological interaction (PPI). In fact, PPI can reveal how inter-regional amygdala communications change dynamically depending on perception of various emotional expressions to recruit different brain networks, compared to the functional interactions it entertains during perception of neutral expressions. We found that for each emotion the amygdala recruited a distinctive and spatially distributed set of structures to interact with. These changes in amygdala connectional patters characterize the dynamic signature prototypical of individual emotion processing, and seemingly represent a neural mechanism that serves to implement the distinctive influence that each emotion exerts on perceptual, cognitive, and motor responses. Besides these differences, all emotions enhanced amygdala functional integration with premotor cortices compared to neutral faces. The present findings thus concur to reconceptualise the structure-function relation between brain-emotion from the traditional one-to-one mapping toward a network-based and dynamic perspective.

  1. Function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

    SciTech Connect

    Nutt, David; Smith, Jeremy C

    2008-10-01

    Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.

  2. Rapid Sampling of Escherichia coli After Changing Oxygen Conditions Reveals Transcriptional Dynamics

    PubMed Central

    von Wulffen, Joachim; Ulmer, Andreas; Jäger, Günter; Sawodny, Oliver; Feuer, Ronny

    2017-01-01

    Escherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression. PMID:28264512

  3. In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

    PubMed Central

    Bajanca, Fernanda; Gonzalez-Perez, Vinicio; Gillespie, Sean J; Beley, Cyriaque; Garcia, Luis; Theveneau, Eric; Sear, Richard P; Hughes, Simon M

    2015-01-01

    Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 PMID:26459831

  4. Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

    PubMed Central

    Grouleff, Julie; Schiøtt, Birgit

    2013-01-01

    The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design. PMID:23776432

  5. Dynamic functional connectivity revealed by resting-state functional near-infrared spectroscopy

    PubMed Central

    Li, Zhen; Liu, Hanli; Liao, Xuhong; Xu, Jingping; Liu, Wenli; Tian, Fenghua; He, Yong; Niu, Haijing

    2015-01-01

    The brain is a complex network with time-varying functional connectivity (FC) and network organization. However, it remains largely unknown whether resting-state fNIRS measurements can be used to characterize dynamic characteristics of intrinsic brain organization. In this study, for the first time, we used the whole-cortical fNIRS time series and a sliding-window correlation approach to demonstrate that fNIRS measurement can be ultimately used to quantify the dynamic characteristics of resting-state brain connectivity. Our results reveal that the fNIRS-derived FC is time-varying, and the variability strength (Q) is correlated negatively with the time-averaged, static FC. Furthermore, the Q values also show significant differences in connectivity between different spatial locations (e.g., intrahemispheric and homotopic connections). The findings are reproducible across both sliding-window lengths and different brain scanning sessions, suggesting that the dynamic characteristics in fNIRS-derived cerebral functional correlation results from true cerebral fluctuation. PMID:26203365

  6. Dynamically regulated miRNA-mRNA networks revealed by exercise

    PubMed Central

    2013-01-01

    Background MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Results Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. Conclusions We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation. PMID:24219008

  7. Dynamically regulated miRNA-mRNA networks revealed by exercise.

    PubMed

    Tonevitsky, Alexander G; Maltseva, Diana V; Abbasi, Asghar; Samatov, Timur R; Sakharov, Dmitry A; Shkurnikov, Maxim U; Lebedev, Alexey E; Galatenko, Vladimir V; Grigoriev, Anatoly I; Northoff, Hinnak

    2013-06-07

    MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.

  8. Polaron dynamics in two types of long oligothiophenes revealed by Q - and X -band ESR measurements

    NASA Astrophysics Data System (ADS)

    Kanemoto, Katsuichi; Furukawa, Ko; Negishi, Nobukazu; Aso, Yoshio; Otsubo, Tetsuo

    2007-10-01

    The polaron dynamics has been investigated through the X - and Q -band ESR measurements for two types of iodine-doped long oligothiophenes, the 20-mer with octyl substituents (o-20T) and the 16-mer with hexyl substituents (h-16T) . o-20T , used as a model compound of conjugated polymers with crystalline grains, gives anisotropic ESR spectra attributed to g anisotropy at low temperatures. The anisotropic spectra are found to be brought by polarons moving within the crystalline grains consisting of parallel chains. The anisotropy is shown to decrease with increasing temperature. This provides definite evidence that the polarons transfer among some grains by the assist of temperature. In contrast, h-16T , used as a model of the polymers with amorphous morphology, gives almost isotropic ESR spectra even in the Q -band measurement. This feature of h-16T is explained to be caused by a rapid interchain transfer of polarons. Spectral simulations performed for obtained spectra reveal that the ESR linewidth in the Q -band measurement is larger than that in the X band for both oligothiophenes. The difference of the linewidth is analyzed by a simplified motional narrowing model in order to draw the information of polaron dynamics. Analyses for o-20T show that the intergrain motion almost follows the variable range hopping model. The interchain motion in h-16T is found to have a much weaker temperature dependence than the intergrain motion in o-20T . This result suggests that the interchain dynamics of h-16T revealed by the ESR technique includes a variety of processes of motion.

  9. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    PubMed Central

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  10. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

    PubMed Central

    Zahari, Muhammad Saddiq; Wu, Xinyan; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Kim, Min-Sik; Fetics, Barry; Philip, Mathew; Barnes, Sheri R.; Godfrey, Beverly; Gabrielson, Edward; Nevo, Erez; Pandey, Akhilesh

    2015-01-01

    Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. PMID:26329039

  11. Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

    PubMed

    Davanture, Marlène; Dumur, Jérôme; Bataillé-Simoneau, Nelly; Campion, Claire; Valot, Benoît; Zivy, Michel; Simoneau, Philippe; Fillinger, Sabine

    2014-07-01

    This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

  12. Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics

    PubMed Central

    Paulo, Joao A.; McAllister, Fiona E.; Everley, Robert A.; Beausoleil, Sean A.; Banks, Alexander S.; Gygi, Steven P.

    2015-01-01

    Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients. PMID:25195567

  13. Proteomic and phosphoproteomic analysis of renal cortex in a salt-load rat model of advanced kidney damage

    PubMed Central

    Jiang, Shaoling; He, Hanchang; Tan, Lishan; Wang, Liangliang; Su, Zhengxiu; Liu, Yufeng; Zhu, Hongguo; Zhang, Menghuan; Hou, Fan Fan; Li, Aiqing

    2016-01-01

    Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex. We found 318 proteins differentially expressed in 5/6Nx group relative to sham group, and 310 proteins significantly changed in response to salt load in 5/6Nx animals. Totally, 1810 unique phosphopeptides corresponding to 550 phosphoproteins were identified. We identified 113 upregulated and 84 downregulated phosphopeptides in 5/6Nx animals relative to sham animals. Salt load induced 78 upregulated and 91 downregulated phosphopeptides in 5/6Nx rats. The differentially expressed phospholproteins are important transporters, structural molecules, and receptors. Protein-protein interaction analysis revealed that the differentially phosphorylated proteins in 5/6Nx group, Polr2a, Srrm1, Gsta2 and Pxn were the most linked. Salt-induced differential phosphoproteins, Myh6, Lmna and Des were the most linked. Altered phosphorylation levels of lamin A and phospholamban were validated. This study will provide new insight into pathogenetic mechanisms of chronic kidney disease and salt sensitivity. PMID:27775022

  14. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

    PubMed

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

    Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond

  15. Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

    PubMed Central

    Bougault, Carole; Aubert-Foucher, Elisabeth; Paumier, Anne; Perrier-Groult, Emeline; Huot, Ludovic; Hot, David; Duterque-Coquillaud, Martine; Mallein-Gerin, Frédéric

    2012-01-01

    Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond

  16. Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis

    PubMed Central

    2010-01-01

    Background Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M). Results TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture. Conclusions This work reports

  17. Dynamic functional connectivity analysis reveals transient states of dysconnectivity in schizophrenia.

    PubMed

    Damaraju, E; Allen, E A; Belger, A; Ford, J M; McEwen, S; Mathalon, D H; Mueller, B A; Pearlson, G D; Potkin, S G; Preda, A; Turner, J A; Vaidya, J G; van Erp, T G; Calhoun, V D

    2014-01-01

    Schizophrenia is a psychotic disorder characterized by functional dysconnectivity or abnormal integration between distant brain regions. Recent functional imaging studies have implicated large-scale thalamo-cortical connectivity as being disrupted in patients. However, observed connectivity differences in schizophrenia have been inconsistent between studies, with reports of hyperconnectivity and hypoconnectivity between the same brain regions. Using resting state eyes-closed functional imaging and independent component analysis on a multi-site data that included 151 schizophrenia patients and 163 age- and gender matched healthy controls, we decomposed the functional brain data into 100 components and identified 47 as functionally relevant intrinsic connectivity networks. We subsequently evaluated group differences in functional network connectivity, both in a static sense, computed as the pairwise Pearson correlations between the full network time courses (5.4 minutes in length), and a dynamic sense, computed using sliding windows (44 s in length) and k-means clustering to characterize five discrete functional connectivity states. Static connectivity analysis revealed that compared to healthy controls, patients show significantly stronger connectivity, i.e., hyperconnectivity, between the thalamus and sensory networks (auditory, motor and visual), as well as reduced connectivity (hypoconnectivity) between sensory networks from all modalities. Dynamic analysis suggests that (1), on average, schizophrenia patients spend much less time than healthy controls in states typified by strong, large-scale connectivity, and (2), that abnormal connectivity patterns are more pronounced during these connectivity states. In particular, states exhibiting cortical-subcortical antagonism (anti-correlations) and strong positive connectivity between sensory networks are those that show the group differences of thalamic hyperconnectivity and sensory hypoconnectivity. Group

  18. Regulation of peroxisome dynamics by phosphorylation.

    PubMed

    Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W; Warscheid, Bettina

    2016-05-01

    Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Revealing the flux: Using processed Husimi maps to visualize dynamics of bound systems and mesoscopic transport

    NASA Astrophysics Data System (ADS)

    Mason, Douglas J.; Borunda, Mario F.; Heller, Eric J.

    2015-04-01

    We elaborate upon the "processed Husimi map" representation for visualizing quantum wave functions using coherent states as a measurement of the local phase space to produce a vector field related to the probability flux. Adapted from the Husimi projection, the processed Husimi map is mathematically related to the flux operator under certain limits but offers a robust and flexible alternative since it can operate away from these limits and in systems that exhibit zero flux. The processed Husimi map is further capable of revealing the full classical dynamics underlying a quantum wave function since it reverse engineers the wave function to yield the underlying classical ray structure. We demonstrate the capabilities of processed Husimi maps on bound systems with and without electromagnetic fields, as well as on open systems on and off resonance, to examine the relationship between closed system eigenstates and mesoscopic transport.

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

    PubMed

    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.

  1. Lagrangian Descriptors: A Method for Revealing Phase Space Structures of General Time Dependent Dynamical Systems

    NASA Astrophysics Data System (ADS)

    Mancho, Ana M.; Wiggins, Stephen; Curbelo, Jezabel; Mendoza, Carolina

    2013-11-01

    Lagrangian descriptors are a recent technique which reveals geometrical structures in phase space and which are valid for aperiodically time dependent dynamical systems. We discuss a general methodology for constructing them and we discuss a ``heuristic argument'' that explains why this method is successful. We support this argument by explicit calculations on a benchmark problem. Several other benchmark examples are considered that allow us to assess the performance of Lagrangian descriptors with both finite time Lyapunov exponents (FTLEs) and finite time averages of certain components of the vector field (``time averages''). In all cases Lagrangian descriptors are shown to be both more accurate and computationally efficient than these methods. We thank CESGA for computing facilities. This research was supported by MINECO grants: MTM2011-26696, I-Math C3-0104, ICMAT Severo Ochoa project SEV-2011-0087, and CSIC grant OCEANTECH. SW acknowledges the support of the ONR (Grant No. N00014-01-1-0769).

  2. Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres.

    PubMed

    Schmidt, Jens C; Zaug, Arthur J; Cech, Thomas R

    2016-08-25

    Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  4. Nanomechanical Behavior of Single Crystalline SiC Nanotubes Revealed by Molecular Dynamics Simulations

    SciTech Connect

    Wang, Zhiguo; Zu, Xiaotao T.; Gao, Fei; Weber, William J.

    2008-11-01

    Molecular dynamics simulations with Tersoff potentials were used to study the response of single crystalline SiC nanotubes under tensile, compressive, torsional, combined tension-torsional and combined compression-torsional strains. The simulation results reveal that the nanotubes deform through bond-stretching and breaking and exhibit brittle properties under uniaxial tensile strain, except for the thinnest nanotube at high temperatures, which fails in a ductile manner. Under uniaxial compressive strain, the SiC nanotubes buckle with two modes, i.e. shell buckling and column buckling, depending on the length of the nanotubes. Under torsional strain, the nanotubes buckle either collapse in the middle region into a dumbbell-like structure for thinner wall thicknesses or fail by bond breakage for the largest wall thickness. Both the tensile failure stress and buckling stress decrease under combined tension-torsional and combined compression-torsional strain, and they decrease with increasing torsional rate under combined loading.

  5. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    PubMed Central

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K.; Temu, Tikira; Oka, Yasuyoshi; Hein, Marco Y.; Nagaraj, Nagarjuna; Long, David T.; Walter, Johannes C.; Hofmann, Kay; Storchova, Zuzana; Cox, Jürgen; Bekker-Jensen, Simon; Mailand, Niels; Mann, Matthias

    2017-01-01

    DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells. PMID:25931565

  6. Proteomics analysis reveals a dynamic diurnal pattern of photosynthesis-related pathways in maize leaves.

    PubMed

    Feng, Dan; Wang, Yanwei; Lu, Tiegang; Zhang, Zhiguo; Han, Xiao

    2017-01-01

    Plant leaves exhibit differentiated patterns of photosynthesis rates under diurnal light regulation. Maize leaves show a single-peak pattern without photoinhibition at midday when the light intensity is maximized. This mechanism contributes to highly efficient photosynthesis in maize leaves. To understand the molecular basis of this process, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics analysis was performed to reveal the dynamic pattern of proteins related to photosynthetic reactions. Steady, single-peak and double-peak protein expression patterns were discovered in maize leaves, and antenna proteins in these leaves displayed a steady pattern. In contrast, the photosystem, carbon fixation and citrate pathways were highly controlled by diurnal light intensity. Most enzymes in the limiting steps of these pathways were major sites of regulation. Thus, maize leaves optimize photosynthesis and carbon fixation outside of light harvesting to adapt to the changes in diurnal light intensity at the protein level.

  7. Sub-structure of laser generated harmonics reveals plasma dynamics of a relativistically oscillating mirror

    SciTech Connect

    Braenzel, J.; Schnürer, M.; Steinke, S.; Priebe, G.; Sandner, W.; Andreev, A.; Platonov, K.

    2013-08-15

    Theoretical and experimental investigations of the dynamics of a relativistically oscillating plasma slab reveal spectral line splitting in laser driven harmonic spectra, leading to double harmonic series. Both series are well characterized with harmonics arising by two fundamental frequencies. While a relativistic oscillation of the critical density drives the harmonic emission, the splitting is a result of an additional acceleration during the laser pulse duration. In comparison with the oscillatory movement, this acceleration is rather weak and can be described by a plasma shock wave driven by the pressure of light. We introduce particle in cell simulations and an analytical model explaining the harmonic line splitting. The derived analytical formula gives direct access between the splitting in the harmonic spectrum and the acceleration of the plasma surface.

  8. Evolutionary dynamics of influenza A nucleoprotein (NP) lineages revealed by large-scale sequence analyses.

    PubMed

    Xu, Jianpeng; Christman, Mary C; Donis, Ruben O; Lu, Guoqing

    2011-12-01

    Influenza A viral nucleoprotein (NP) plays a critical role in virus replication and host adaptation, however, the underlying molecular evolutionary dynamics of NP lineages are less well-understood. In this study, large-scale analyses of 5094 NP nucleotide sequences revealed eight distinct evolutionary lineages, including three host-specific lineages (human, classical swine and equine), two cross-host lineages (Eurasian avian-like swine and swine-origin human pandemic H1N1 2009) and three geographically isolated avian lineages (Eurasian, North American and Oceanian). The average nucleotide substitution rate of the NP lineages was estimated to be 2.4 × 10(-3) substitutions per site per year, with the highest value observed in pandemic H1N1 2009 (3.4 × 10(-3)) and the lowest in equine (0.9 × 10(-3)). The estimated time of most recent common ancestor (TMRCA) for each lineage demonstrated that the earliest human lineage was derived around 1906, and the latest pandemic H1N1 2009 lineage dated back to December 17, 2008. A marked time gap was found between the times when the viruses emerged and were first sampled, suggesting the crucial role for long-term surveillance of newly emerging viruses. The selection analyses showed that human lineage had six positive selection sites, whereas pandemic H1N1 2009, classical swine, Eurasian avian and Eurasian swine had only one or two sites. Protein structure analyses revealed several positive selection sites located in epitope regions or host adaptation regions, indicating strong adaptation to host immune system pressures in influenza viruses. Along with previous studies, this study provides new insights into the evolutionary dynamics of influenza A NP lineages. Further lineage analyses of other gene segments will allow better understanding of influenza A virus evolution and assist in the improvement of global influenza surveillance.

  9. Constraints on Genome Dynamics Revealed from Gene Distribution among the Ralstonia solanacearum Species

    PubMed Central

    Lefeuvre, Pierre; Cellier, Gilles; Remenant, Benoît; Chiroleu, Frédéric; Prior, Philippe

    2013-01-01

    Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens. PMID:23723974

  10. Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

    PubMed Central

    Lee, Dong-Hyun; Goodarzi, Aaron A; Adelmant, Guillaume O; Pan, Yunfeng; Jeggo, Penelope A; Marto, Jarrod A; Chowdhury, Dipanjan

    2012-01-01

    Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response. PMID:22491012

  11. Postnatal changes in the growth dynamics of the human face revealed from bone modelling patterns

    PubMed Central

    Martinez-Maza, Cayetana; Rosas, Antonio; Nieto-Díaz, Manuel

    2013-01-01

    Human skull morphology results from complex processes that involve the coordinated growth and interaction of its skeletal components to keep a functional and structural balance. Previous histological works have studied the growth of different craniofacial regions and their relationship to functional spaces in humans up to 14 years old. Nevertheless, how the growth dynamics of the facial skeleton and the mandible are related and how this relationship changes through the late ontogeny remain poorly understood. To approach these two questions, we have compared the bone modelling activities of the craniofacial skeleton from a sample of subadult and adult humans. In this study, we have established for the first time the bone modelling pattern of the face and the mandible from adult humans. Our analyses reveal a patchy distribution of the bone modelling fields (overemphasized by the presence of surface islands with no histological information) reflecting the complex growth dynamics associated to the individual morphology. Subadult and adult specimens show important differences in the bone modelling patterns of the anterior region of the facial skeleton and the posterior region of the mandible. These differences indicate developmental changes in the growth directions of the whole craniofacial complex, from a predominantly downward growth in subadults that turns to a forward growth observed in the adult craniofacial skeleton. We hypothesize that these ontogenetic changes would respond to the physiological and physical requirements to enlarge the oral and nasal cavities once maturation of the brain and the closure of the cranial sutures have taken place during craniofacial development. PMID:23819603

  12. Health trajectories reveal the dynamic contributions of host genetic resistance and tolerance to infection outcome

    PubMed Central

    Lough, Graham; Kyriazakis, Ilias; Bergmann, Silke; Lengeling, Andreas; Doeschl-Wilson, Andrea B.

    2015-01-01

    Resistance and tolerance are two alternative strategies hosts can adopt to survive infections. Both strategies may be genetically controlled. To date, the relative contribution of resistance and tolerance to infection outcome is poorly understood. Here, we use a bioluminescent Listeria monocytogenes (Lm) infection challenge model to study the genetic determination and dynamic contributions of host resistance and tolerance to listeriosis in four genetically diverse mouse strains. Using conventional statistical analyses, we detect significant genetic variation in both resistance and tolerance, but cannot capture the time-dependent relative importance of either host strategy. We overcome these limitations through the development of novel statistical tools to analyse individual infection trajectories portraying simultaneous changes in infection severity and health. Based on these tools, early expression of resistance followed by expression of tolerance emerge as important hallmarks for surviving Lm infections. Our trajectory analysis further reveals that survivors and non-survivors follow distinct infection paths (which are also genetically determined) and provides new survival thresholds as objective endpoints in infection experiments. Future studies may use trajectories as novel traits for mapping and identifying genes that control infection dynamics and outcome. A Matlab script for user-friendly trajectory analysis is provided. PMID:26582028

  13. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis

    NASA Astrophysics Data System (ADS)

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-01

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  14. Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

    PubMed

    Kong, Muwen; Liu, Lili; Chen, Xuejing; Driscoll, Katherine I; Mao, Peng; Böhm, Stefanie; Kad, Neil M; Watkins, Simon C; Bernstein, Kara A; Wyrick, John J; Min, Jung-Hyun; Van Houten, Bennett

    2016-10-20

    Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of β-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Multifractal analysis of Barkhausen noise reveals the dynamic nature of criticality at hysteresis loop

    NASA Astrophysics Data System (ADS)

    Tadić, Bosiljka

    2016-06-01

    The field-driven magnetisation reversal processes in disordered systems exhibit a collective behaviour that is manifested in the scale-invariance of avalanches, closely related to underlying dynamical mechanisms. Using the multifractal time series analysis, we study the structure of fluctuations at different scales in the accompanying Barkhausen noise. The stochastic signal represents the magnetisation discontinuities along the hysteresis loop of a three-dimensional random field Ising model simulated for varied disorder strength and driving rates. The analysis of the spectrum of the generalised Hurst exponents reveals that the dominant segments of the signal with large fluctuations represent two distinct classes of stochastic processes in weak and strong pinning regimes. Furthermore, in the weak pinning regime, the part of the signal originating from the beginning of the hysteresis loop has a different multifractal spectrum than the signal near the coercive field. The enhanced fluctuations (primarily in the central part of the hysteresis loop) for increased driving rate and larger system size, lead to a further broadening of the spectrum. The analysed Barkhausen signals are also shown to exhibit temporal correlations and power-law distributions of the magnetisation discontinuity and avalanche sizes, in agreement with previous studies. The multifractal properties of Barkhausen noise describe the dynamical state of domains and precisely discriminate the weak pinning, permitting the motion of individual walls, from the mechanisms occurring in strongly disordered systems.

  16. Dynamic changes in brewing yeast cells in culture revealed by statistical analyses of yeast morphological data.

    PubMed

    Ohnuki, Shinsuke; Enomoto, Kenichi; Yoshimoto, Hiroyuki; Ohya, Yoshikazu

    2014-03-01

    The vitality of brewing yeasts has been used to monitor their physiological state during fermentation. To investigate the fermentation process, we used the image processing software, CalMorph, which generates morphological data on yeast mother cells and bud shape, nuclear shape and location, and actin distribution. We found that 248 parameters changed significantly during fermentation. Successive use of principal component analysis (PCA) revealed several important features of yeast, providing insight into the dynamic changes in the yeast population. First, PCA indicated that much of the observed variability in the experiment was summarized in just two components: a change with a peak and a change over time. Second, PCA indicated the independent and important morphological features responsible for dynamic changes: budding ratio, nucleus position, neck position, and actin organization. Thus, the large amount of data provided by imaging analysis can be used to monitor the fermentation processes involved in beer and bioethanol production. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

  18. Application of Lifeact Reveals F-Actin Dynamics in Arabidopsis thaliana and the Liverwort, Marchantia polymorpha

    PubMed Central

    Era, Atsuko; Tominaga, Motoki; Ebine, Kazuo; Awai, Chie; Saito, Chieko; Ishizaki, Kimitsune; Yamato, Katsuyuki T.; Kohchi, Takayuki; Nakano, Akihiko; Ueda, Takashi

    2009-01-01

    Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact–Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact–Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages. PMID:19369273

  19. Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha.

    PubMed

    Era, Atsuko; Tominaga, Motoki; Ebine, Kazuo; Awai, Chie; Saito, Chieko; Ishizaki, Kimitsune; Yamato, Katsuyuki T; Kohchi, Takayuki; Nakano, Akihiko; Ueda, Takashi

    2009-06-01

    Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact-Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact-Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.

  20. Dynamics of Primate Oculomotor Plant Revealed by Effects of Abducens Microstimulation

    PubMed Central

    Anderson, Sean R.; Porrill, John; Sklavos, Sokratis; Gandhi, Neeraj J.; Sparks, David L.; Dean, Paul

    2009-01-01

    Despite their importance for deciphering oculomotor commands, the mechanics of the extraocular muscles and orbital tissues (oculomotor plant) are poorly understood. In particular, the significance of plant nonlinearities is uncertain. Here primate plant dynamics were investigated by measuring the eye movements produced by stimulating the abducens nucleus with brief pulse trains of varying frequency. Statistical analysis of these movements indicated that the effects of stimulation lasted about 40 ms after the final pulse, after which the eye returned passively toward its position before stimulation. Behavior during the passive phase could be approximated by a linear plant model, corresponding to Voigt elements in series, with properties independent of initial eye position. In contrast, behavior during the stimulation phase revealed a sigmoidal relation between stimulation frequency and estimated steady-state tetanic tension, together with a frequency-dependent rate of tension increase, that appeared very similar to the nonlinearities previously found for isometric-force production in primate lateral rectus muscle. These results suggest that the dynamics of the oculomotor plant have an approximately linear component related to steady-state viscoelasticity and a nonlinear component related to changes in muscle activation. The latter may in part account for the nonlinear relations observed between eye-movement parameters and single-unit firing patterns in the abducens nucleus. These findings point to the importance of recruitment as a simplifying factor for motor control with nonlinear plants. PMID:19297512

  1. Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

    PubMed Central

    Marzi, Matteo J.; Ghini, Francesco; Cerruti, Benedetta; de Pretis, Stefano; Bonetti, Paola; Giacomelli, Chiara; Gorski, Marcin M.; Kress, Theresia; Pelizzola, Mattia; Muller, Heiko; Amati, Bruno; Nicassio, Francesco

    2016-01-01

    The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2 > 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2 = 4–14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions. PMID:26821571

  2. Revealing dynamic processes of materials in liquids using liquid cell transmission electron microscopy.

    PubMed

    Niu, Kai-Yang; Liao, Hong-Gang; Zheng, Haimei

    2012-12-20

    The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

  3. Sarcomere dynamics in single myocardial cells as revealed by high-resolution light diffractometry.

    PubMed

    Leung, A F

    1983-08-01

    A specially designed diffractometer with a high spatial and temporal resolution recorded the diffraction of a laser beam by single enzymatically isolated myocardial cells. The fine structures within the first-order diffraction were resolved and each structure was interpreted as the diffraction from a group of sarcomeres of nearly equal length. During activation of the cell dynamics of each discrete group of sarcomeres was uniform and independent of the other groups. However, a small nonuniform component in the sarcomere dynamics was observed and attributed to the coupling between the shortening tension and the radial stress resulting from the expansion of the myofibrillar cross-section. The time-course of the diffraction fine structures during contractile activity revealed (1) the period of the contraction-relaxation cycle, (2) the latent period, (3) the shortening and relengthening speeds and (4) the variation in the line width and intensity of the fine structure. Measurements showed that the latent period was dependent on the free Ca2+ of the cell's bathing solution while the initial shortening speed was not. The diffraction line width and intensity of the shortening cell were explained by the grating model.

  4. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.

    PubMed

    Trapnell, Cole; Cacchiarelli, Davide; Grimsby, Jonna; Pokharel, Prapti; Li, Shuqiang; Morse, Michael; Lennon, Niall J; Livak, Kenneth J; Mikkelsen, Tarjei S; Rinn, John L

    2014-04-01

    Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.

  5. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

    PubMed

    Hertig, Samuel; Latorraca, Naomi R; Dror, Ron O

    2016-06-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

  6. Suicide Gene-Engineered Stromal Cells Reveal a Dynamic Regulation of Cancer Metastasis.

    PubMed

    Shen, Keyue; Luk, Samantha; Elman, Jessica; Murray, Ryan; Mukundan, Shilpaa; Parekkadan, Biju

    2016-02-19

    Cancer-associated fibroblasts (CAFs) are a major cancer-promoting component in the tumor microenvironment (TME). The dynamic role of human CAFs in cancer progression has been ill-defined because human CAFs lack a unique marker needed for a cell-specific, promoter-driven knockout model. Here, we developed an engineered human CAF cell line with an inducible suicide gene to enable selective in vivo elimination of human CAFs at different stages of xenograft tumor development, effectively circumventing the challenge of targeting a cell-specific marker. Suicide-engineered CAFs were highly sensitive to apoptosis induction in vitro and in vivo by the addition of a simple small molecule inducer. Selection of timepoints for targeted CAF apoptosis in vivo during the progression of a human breast cancer xenograft model was guided by a bi-phasic host cytokine response that peaked at early timepoints after tumor implantation. Remarkably, we observed that the selective apoptosis of CAFs at these early timepoints did not affect primary tumor growth, but instead increased the presence of tumor-associated macrophages and the metastatic spread of breast cancer cells to the lung and bone. The study revealed a dynamic relationship between CAFs and cancer metastasis that has counter-intuitive ramifications for CAF-targeted therapy.

  7. DNA methylation dynamics during intestinal stem cell differentiation reveals enhancers driving gene expression in the villus

    PubMed Central

    2013-01-01

    Background DNA methylation is of pivotal importance during development. Previous genome-wide studies identified numerous differentially methylated regions upon differentiation of stem cells, many of them associated with transcriptional start sites. Results We present the first genome-wide, single-base-resolution view into DNA methylation dynamics during differentiation of a mammalian epithelial stem cell: the mouse small intestinal Lgr5+ stem cell. Very little change was observed at transcriptional start sites and our data suggest that differentiation-related genes are already primed for expression in the stem cell. Genome-wide, only 50 differentially methylated regions were identified. Almost all of these loci represent enhancers driving gene expression in the differentiated part of the small intestine. Finally, we show that binding of the transcription factor Tcf4 correlates with hypo-methylation and demonstrate that Tcf4 is one of the factors contributing to formation of differentially methylated regions. Conclusions Our results reveal limited DNA methylation dynamics during small intestine stem cell differentiation and an impact of transcription factor binding on shaping the DNA methylation landscape during differentiation of stem cells in vivo. PMID:23714178

  8. ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

    PubMed Central

    De Col, Valentina; Fuchs, Philippe; Nietzel, Thomas; Elsässer, Marlene; Voon, Chia Pao; Candeo, Alessia; Seeliger, Ingo; Fricker, Mark D; Grefen, Christopher; Møller, Ian Max; Bassi, Andrea; Lim, Boon Leong; Zancani, Marco; Meyer, Andreas J; Costa, Alex; Wagner, Stephan; Schwarzländer, Markus

    2017-01-01

    Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant. DOI: http://dx.doi.org/10.7554/eLife.26770.001 PMID:28716182

  9. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

    PubMed Central

    Hertig, Samuel

    2016-01-01

    Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest. PMID:27285999

  10. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  11. Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea

    PubMed Central

    Parsons, Rachel J; Breitbart, Mya; Lomas, Michael W; Carlson, Craig A

    2012-01-01

    There are an estimated 1030 virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean

  12. Ocean time-series reveals recurring seasonal patterns of virioplankton dynamics in the northwestern Sargasso Sea.

    PubMed

    Parsons, Rachel J; Breitbart, Mya; Lomas, Michael W; Carlson, Craig A

    2012-02-01

    There are an estimated 10(30) virioplankton in the world oceans, the majority of which are phages (viruses that infect bacteria). Marine phages encompass enormous genetic diversity, affect biogeochemical cycling of elements, and partially control aspects of prokaryotic production and diversity. Despite their importance, there is a paucity of data describing virioplankton distributions over time and depth in oceanic systems. A decade of high-resolution time-series data collected from the upper 300 m in the northwestern Sargasso Sea revealed recurring temporal and vertical patterns of virioplankton abundance in unprecedented detail. An annual virioplankton maximum developed between 60 and 100 m during periods of summer stratification and eroded during winter convective mixing. The timing and vertical positioning of this seasonal pattern was related to variability in water column stability and the dynamics of specific picophytoplankton and heterotrophic bacterioplankton lineages. Between 60 and 100 m, virioplankton abundance was negatively correlated to the dominant heterotrophic bacterioplankton lineage SAR11, as well as the less abundant picophytoplankton, Synechococcus. In contrast, virioplankton abundance was positively correlated to the dominant picophytoplankton lineage Prochlorococcus, and the less abundant alpha-proteobacteria, Rhodobacteraceae. Seasonally, virioplankton abundances were highly synchronous with Prochlorococcus distributions and the virioplankton to Prochlorococcus ratio remained remarkably constant during periods of water column stratification. The data suggest that a significant fraction of viruses in the mid-euphotic zone of the subtropical gyres may be cyanophages and patterns in their abundance are largely determined by Prochlorococcus dynamics in response to water column stability. This high-resolution, decadal survey of virioplankton abundance provides insight into the possible controls of virioplankton dynamics in the open ocean.

  13. DNA from lake sediments reveals the long-term dynamics and diversity of Synechococcus assemblages

    NASA Astrophysics Data System (ADS)

    Domaizon, I.; Savichtcheva, O.; Debroas, D.; Arnaud, F.; Villar, C.; Pignol, C.; Alric, B.; Perga, M. E.

    2013-02-01

    While picocyanobacteria (PC) are important actors in carbon and nutrient cycles in aquatic systems, factors controlling their interannual dynamics and diversity are poorly known due to the general lack of long-term monitoring surveys. This study intended to fill this gap by applying a DNA-based paleolimnological approach to sediment records from a deep subalpine lake that has experienced dramatic changes in environmental conditions during the last century (eutrophication, re-oligotrophication and large-scale climate changes). We particularly investigated the long-term (100 yr) diversity and dynamics of Synechococcus, PC that have presumably been affected by both the lake trophic status changes and global warming. The lake's morphological and environmental conditions provided ideal conditions for DNA preservation in the sediment archives. Generalised additive models applied to quantitative PCR (qPCR) results highlighted that an increase in summer temperature could have a significant positive impact on the relative abundance of Synechococcus (fraction of Synechococcus in total cyanobacteria). The diversity of Synechococcus in Lake Bourget was studied by phylogenetic analyses of the 16S rRNA gene and internal transcribed spacer (ITS). Up to 23 different OTUs (based on 16S rRNA), which fell into various cosmopolitan or endemic clusters, were identified in samples from the past 100 yr. Moreover, study of the ITS revealed a higher diversity within the major 16S rRNA-defined OTUs. Changes in PC diversity were related to the lake's trophic status. Overall, qPCR and sequencing results showed that environmental changes (here, in temperature and phosphorus concentration) affected Synechococcus community dynamics and structure, translating into changes in genotype composition. These results also helped to re-evaluate the geographical distribution of some Synechococcus clusters. Providing such novel insights into the long-term history of an important group of primary producers

  14. DNA from lake sediments reveals the long-term dynamics and diversity of Synechococcus assemblages

    NASA Astrophysics Data System (ADS)

    Domaizon, I.; Savichtcheva, O.; Debroas, D.; Arnaud, F.; Villar, C.; Pignol, C.; Alric, B.; Perga, M. E.

    2013-06-01

    While picocyanobacteria (PC) are important actors in carbon and nutrient cycles in aquatic systems, factors controlling their interannual dynamics and diversity are poorly known due to the general lack of long-term monitoring surveys. This study intended to fill this gap by applying a DNA-based paleolimnological approach to sediment records from a deep subalpine lake that has experienced dramatic changes in environmental conditions during the last century (eutrophication, re-oligotrophication and large-scale climate changes). In particular, we investigated the long-term (100 yr) diversity and dynamics of Synechococcus,, PC that have presumably been affected by both the lake trophic status changes and global warming. The lake's morphological and environmental conditions provided the ideal conditions for DNA preservation in the sediment archives. Generalised additive models applied to quantitative PCR (qPCR; quantitative Polymerase Chain Reaction) results highlighted that an increase in summer temperature could have a significant positive impact on the relative abundance of Synechococcus, (fraction of Synechococcus, in total cyanobacteria). The diversity of Synechococcus, in Lake Bourget was studied by phylogenetic analyses of the 16S rRNA gene and the following internally transcribed spacer (ITS). Up to 23 different OTUs (based on 16S rRNA), which fell into various cosmopolitan or endemic clusters, were identified in samples from the past 100 yr. Moreover, the study of ITS revealed a higher diversity within the major 16S rRNA-defined OTUs. Changes in PC diversity were related to the lake's trophic status. Overall, qPCR and sequencing results showed that environmental changes (in temperature and phosphorus concentration) affected Synechococcus, community dynamics and structure, translating into changes in genotype composition. These results also helped to re-evaluate the geographical distribution of some Synechococcus, clusters. Providing such novel insights into the

  15. Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family.

    PubMed

    Levine, Mia T; McCoy, Connor; Vermaak, Danielle; Lee, Yuh Chwen G; Hiatt, Mary Alice; Matsen, Frederick A; Malik, Harmit S

    2012-01-01

    Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.

  16. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

    PubMed Central

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

    2016-01-01

    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

  17. Revealing nonergodic dynamics in living cells from a single particle trajectory

    NASA Astrophysics Data System (ADS)

    Lanoiselée, Yann; Grebenkov, Denis S.

    2016-05-01

    We propose the improved ergodicity and mixing estimators to identify nonergodic dynamics from a single particle trajectory. The estimators are based on the time-averaged characteristic function of the increments and can thus capture additional information on the process as compared to the conventional time-averaged mean-square displacement. The estimators are first investigated and validated for several models of anomalous diffusion, such as ergodic fractional Brownian motion and diffusion on percolating clusters, and nonergodic continuous-time random walks and scaled Brownian motion. The estimators are then applied to two sets of earlier published trajectories of mRNA molecules inside live Escherichia coli cells and of Kv2.1 potassium channels in the plasma membrane. These statistical tests did not reveal nonergodic features in the former set, while some trajectories of the latter set could be classified as nonergodic. Time averages along such trajectories are thus not representative and may be strongly misleading. Since the estimators do not rely on ensemble averages, the nonergodic features can be revealed separately for each trajectory, providing a more flexible and reliable analysis of single-particle tracking experiments in microbiology.

  18. Dynamical modelling of prostaglandin signalling in platelets reveals individual receptor contributions and feedback properties.

    PubMed

    Mischnik, Marcel; Hubertus, Katharina; Geiger, Jörg; Dandekar, Thomas; Timmer, Jens

    2013-10-01

    Prostaglandins are the key-players in diminishing platelet function. They exert their effects via a variety of surface receptors that are linked to the cAMP/PKA-signalling cascade. However, less is known about the quantitative impact of the individual receptors on the underlying pathway. We present here a comprehensive ordinary differential equation-based model of the platelet cAMP pathway, including the four prostaglandin receptors IP, DP1, EP3 and EP4, the ADP receptor P2Y12, a detailed PKA-module as well as downstream-targets. Parameter estimation along with a comprehensive combination of time-course and dose-response measurements revealed the individual quantitative role of each receptor in elevating or decreasing pathway activity. A comparison of the two inhibiting receptors EP3 and P2Y12 exhibited a greater signalling strength of the EP3 receptor with implications for antithrombotic treatment. Furthermore, analysis of different model topologies revealed a direct influence of PKA on adenylate cyclase, reducing its maximum catalytic speed. Finally, we show here for the first time the dynamic behaviour of VASP-phosphorylation, which is commonly used as a marker for platelet-inhibition. We validate our model by comparing it to further experimental data.

  19. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

    PubMed Central

    Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

    2011-01-01

    Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

  20. Mechanical properties of a complete microtubule revealed through molecular dynamics simulation.

    PubMed

    Wells, David B; Aksimentiev, Aleksei

    2010-07-21

    Microtubules (MTs) are the largest type of cellular filament, essential in processes ranging from mitosis and meiosis to flagellar motility. Many of the processes depend critically on the mechanical properties of the MT, but the elastic moduli, notably the Young's modulus, are not directly revealed in experiment, which instead measures either flexural rigidity or response to radial deformation. Molecular dynamics (MD) is a method that allows the mechanical properties of single biomolecules to be investigated through computation. Typically, MD requires an atomic resolution structure of the molecule, which is unavailable for many systems, including MTs. By combining structural information from cryo-electron microscopy and electron crystallography, we have constructed an all-atom model of a complete MT and used MD to determine its mechanical properties. The simulations revealed nonlinear axial stress-strain behavior featuring a pronounced softening under extension, a possible plastic deformation transition under radial compression, and a distinct asymmetry in response to the two senses of twist. This work demonstrates the possibility of combining different levels of structural information to produce all-atom models suitable for quantitative MD simulations, which extends the range of systems amenable to the MD method and should enable exciting advances in our microscopic knowledge of biology. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors

    NASA Astrophysics Data System (ADS)

    Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

    2015-05-01

    In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

  2. Dynamics of HIV-Containing Compartments in Macrophages Reveal Sequestration of Virions and Transient Surface Connections

    PubMed Central

    Gaudin, Raphaël; Berre, Stefano; Cunha de Alencar, Bruna; Decalf, Jérémie; Schindler, Michael; Gobert, François-Xavier; Jouve, Mabel; Benaroch, Philippe

    2013-01-01

    During HIV pathogenesis, infected macrophages behave as “viral reservoirs” that accumulate and retain virions within dedicated internal Virus-Containing Compartments (VCCs). The nature of VCCs remains ill characterized and controversial. Using wild-type HIV-1 and a replication-competent HIV-1 carrying GFP internal to the Gag precursor, we analyzed the biogenesis and evolution of VCCs in primary human macrophages. VCCs appear roughly 14 hours after viral protein synthesis is detected, initially contain few motile viral particles, and then mature to fill up with virions that become packed and immobile. The amount of intracellular Gag, the proportion of dense VCCs, and the density of viral particles in their lumen increased with time post-infection. In contrast, the secretion of virions, their infectivity and their transmission to T cells decreased overtime, suggesting that HIV-infected macrophages tend to pack and retain newly formed virions into dense compartments. A minor proportion of VCCs remains connected to the plasma membrane overtime. Surprisingly, live cell imaging combined with correlative light and electron microscopy revealed that such connections can be transient, highlighting their dynamic nature. Together, our results shed light on the late phases of the HIV-1 cycle and reveal some of its macrophage specific features. PMID:23922713

  3. Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors.

    PubMed

    Sala, Adrien; Shoaib, Muhammad; Anufrieva, Olga; Mutharasu, Gnanavel; Jahan Hoque, Rawnak; Yli-Harja, Olli; Kandhavelu, Meenakshisundaram

    2015-05-19

    In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

  4. Event-Scale Morphodynamics and Sediment Sorting in a Dynamic Braided River Revealed by TLS

    NASA Astrophysics Data System (ADS)

    Vericat, D.; Brasington, J.

    2008-12-01

    In the last decade, advances in topographic survey and digital elevation modelling have enabled a revolution in the study of fluvial morphodynamics. Despite this recent progress, our understanding of braided river dynamics remains limited by the time-space scale of studies. Hindered by high labour and flight costs, together with slow ground-based survey methods, studies to date have focused either on event-scale dynamics of morphological units (Ferguson and Ashworth, 1992; Lane et al., 1995; Milan et al., 2007) or seasonal-annual dynamics of larger system-scale reaches (sensu Lane, 2006; e.g., Brasington et al., 2003; Lane et al., 2003). Terrestrial Laser Scanning technology offers the potential to acquire rapidly, reach-scale datasets which record topographic information at the resolution of bed grain-scale upwards. However, as yet, no detailed 3d datasets exist that reveal the system-scale evolution of a braided river through a continuous sequence of floods. Such data are urgently required to address unresolved and fundamental questions concerning the controls and behaviour of braided rivers and are also needed to validate morphodynamic simulation models (Brasington and Richards, 2007). Our recent wok has demonstrated that TLS can be applied to recover centimetre-scale channel morphology, maps of particle size, sorting, packing and floodplain roughness (Brasington et al., 2007, 2008; Antonarakis, 2008a,b; Hodge et al., in review). This potential is illustrated by the results obtained in a field study conducted in January 2008. This used TLS to monitor the evolution of channel morphology and develop methods to derive models of bed roughness and facies in a small 500 x 300 m reach of the actively braided Rees River, New Zealand. Fieldwork comprised repeat surveys before and after 3 competent events, combining laser scans from eight positions with bathymetric data obtained by RTK GPS. The resulting point clouds incorporated between 48-110 million survey points, with

  5. Gas Dynamics and Outflow in the Barred Starburst Galaxy NGC 1808 Revealed with ALMA

    NASA Astrophysics Data System (ADS)

    Salak, Dragan; Nakai, Naomasa; Hatakeyama, Takuya; Miyamoto, Yusuke

    2016-05-01

    NGC 1808 is a nearby barred starburst galaxy with an outflow from the nuclear region. To study the inflow and outflow processes related to star formation and dynamical evolution of the galaxy, we have carried out 12CO (J=1-0) mapping observations of the central r ˜ 4 kpc of NGC 1808 using the Atacama Large Millimeter/submillimeter Array. Four distinct components of molecular gas are revealed at high spatial resolution of 2″ (˜100 pc): (1) a compact (r < 200 pc) circumnuclear disk (CND), (2) r ˜ 500 pc ring, (3) gas-rich galactic bar, and (4) spiral arms. Basic geometric and kinematic parameters are derived for the central 1 kpc region using tilted-ring modeling. The derived rotation curve reveals multiple mass components that include (1) a stellar bulge, (2) a nuclear bar and molecular CND, and (3) an unresolved massive (˜107 M ⊙) core. Two systemic velocities, 998 km s-1 for the CND and 964 km s-1 for the 500 pc ring, are revealed, indicating a kinematic offset. The pattern speed of the primary bar, derived by using a cloud-orbit model, is 56 ± 11 km s-1 kpc-1. Noncircular motions are detected associated with a nuclear spiral pattern and outflow in the central 1 kpc region. The ratio of the mass outflow rate to the star formation rate is {\\dot{M}}{out}/{SFR}˜ 0.2 in the case of optically thin CO (1-0) emission in the outflow, suggesting low efficiency of star formation quenching.

  6. Mechanically untying a protein slipknot: multiple pathways revealed by force spectroscopy and steered molecular dynamics simulations.

    PubMed

    He, Chengzhi; Genchev, Georgi Z; Lu, Hui; Li, Hongbin

    2012-06-27

    Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.

  7. Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations

    PubMed Central

    He, Chengzhi; Genchev, Georgi Z.; Lu, Hui; Li, Hongbin

    2013-01-01

    Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechancial unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie, and the polypeptide chain to completely extend. These distinct unfolding pathways proceed either via a two-state or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus provding a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechancial unfolding pathway of AFV3-109, and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding. PMID:22626004

  8. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids

    PubMed Central

    Guerreiro, Joana F.; Mira, Nuno P.; Santos, Aline X. S.; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the “Npr1-family” of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis. PMID:28747907

  9. Membrane Phosphoproteomics of Yeast Early Response to Acetic Acid: Role of Hrk1 Kinase and Lipid Biosynthetic Pathways, in Particular Sphingolipids.

    PubMed

    Guerreiro, Joana F; Mira, Nuno P; Santos, Aline X S; Riezman, Howard; Sá-Correia, Isabel

    2017-01-01

    Saccharomyces cerevisiae response and tolerance to acetic acid is critical in industrial biotechnology and in acidic food and beverages preservation. The HRK1 gene, encoding a protein kinase of unknown function belonging to the "Npr1-family" of kinases known to be involved in the regulation of plasma membrane transporters, is an important determinant of acetic acid tolerance. This study was performed to identify the alterations occurring in yeast membrane phosphoproteome profile during the adaptive early response to acetic acid stress (following 1 h of exposure to a sub-lethal inhibitory concentration; 50 mM at pH 4.0) and the effect of HRK1 expression on the phosphoproteome. Results from mass spectrometry analysis following the prefractionation and specific enrichment of phosphorylated peptides using TiO2 beads highlight the contribution of processes related with translation, protein folding and processing, transport, and cellular homeostasis in yeast response to acetic acid stress, with particular relevance for changes in phosphorylation of transport-related proteins, found to be highly dependent on the Hrk1 kinase. Twenty different phosphoproteins known to be involved in lipid and sterol metabolism were found to be differently phosphorylated in response to acetic acid stress, including several phosphopeptides that had not previously been described as being phosphorylated. The suggested occurrence of cellular lipid composition remodeling during the short term yeast response to acetic acid was confirmed: Hrk1 kinase-independent reduction in phytoceramide levels and a reduction in phosphatidylcholine and phosphatidylinositol levels under acetic acid stress in the more susceptible hrk1Δ strain were revealed by a lipidomic analysis.

  10. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  11. Reduction in Dynamic Visual Acuity Reveals Gaze Control Changes Following Spaceflight

    NASA Technical Reports Server (NTRS)

    Peters, Brian T.; Brady, Rachel A.; Miller, Chris; Lawrence, Emily L.; Mulavara Ajitkumar P.; Bloomberg, Jacob J.

    2010-01-01

    INTRODUCTION: Exposure to microgravity causes adaptive changes in eye-head coordination that can lead to altered gaze control. This could affect postflight visual acuity during head and body motion. The goal of this study was to characterize changes in dynamic visual acuity after long-duration spaceflight. METHODS: Dynamic Visual Acuity (DVA) data from 14 astro/cosmonauts were collected after long-duration (6 months) spaceflight. The difference in acuity between seated and walking conditions provided a metric of change in the subjects ability to maintain gaze fixation during self-motion. In each condition, a psychophysical threshold detection algorithm was used to display Landolt ring optotypes at a size that was near each subject s acuity threshold. Verbal responses regarding the orientation of the gap were recorded as the optotypes appeared sequentially on a computer display 4 meters away. During the walking trials, subjects walked at 6.4 km/h on a motorized treadmill. RESULTS: A decrement in mean postflight DVA was found, with mean values returning to baseline within 1 week. The population mean showed a consistent improvement in DVA performance, but it was accompanied by high variability. A closer examination of the individual subject s recovery curves revealed that many did not follow a pattern of continuous improvement with each passing day. When adjusted on the basis of previous long-duration flight experience, the population mean shows a "bounce" in the re-adaptation curve. CONCLUSION: Gaze control during self-motion is altered following long-duration spaceflight and changes in postflight DVA performance indicate that vestibular re-adaptation may be more complex than a gradual return to normal.

  12. Identifying habitats at risk: simple models can reveal complex ecosystem dynamics.

    PubMed

    Maxwell, Paul S; Pitt, Kylie A; Olds, Andrew D; Rissik, David; Connolly, Rod M

    2015-03-01

    The relationship between ecological impact and ecosystem structure is often strongly nonlinear, so that small increases in impact levels can cause a disproportionately large response in ecosystem structure. Nonlinear ecosystem responses can be difficult to predict because locally relevant data sets can be difficult or impossible to obtain. Bayesian networks (BN) are an emerging tool that can help managers to define ecosystem relationships using a range of data types from comprehensive quantitative data sets to expert opinion. We show how a simple BN can reveal nonlinear dynamics in seagrass ecosystems using ecological relationships sourced from the literature. We first developed a conceptual diagram by cataloguing the ecological responses of seagrasses to a range of drivers and impacts. We used the conceptual diagram to develop a BN populated with values sourced from published studies. We then applied the BN to show that the amount of initial seagrass biomass has a mitigating effect on the level of impact a meadow can withstand without loss, and that meadow recovery can often require disproportionately large improvements in impact levels. This mitigating effect resulted in the middle ranges of impact levels having a wide likelihood of seagrass presence, a situation known as bistability. Finally, we applied the model in a case study to identify the risk of loss and the likelihood of recovery for the conservation and management of seagrass meadows in Moreton Bay, Queensland, Australia. We used the model to predict the likelihood of bistability in 23 locations in the Bay. The model predicted bistability in seven locations, most of which have experienced seagrass loss at some stage in the past 25 years providing essential information for potential future restoration efforts. Our results demonstrate the capacity of simple, flexible modeling tools to facilitate collation and synthesis of disparate information. This approach can be adopted in the initial stages of

  13. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    PubMed

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

  14. The Dynamics of Sea Straits Reveals Large-Scale Modes of Variability

    NASA Astrophysics Data System (ADS)

    Rubino, Angelo; Androsov, Alexey; Zanchettin, Davide; Voltzinger, Naum

    2016-04-01

    Using a very high resolution 3D numerical model we investigate the tidal dynamics in the Strait of Messina. We show that different stratifications at the southern boundaries, consistent with observed stratifications in the Ionian approaches to the Strait, yield different mean sea level heights. On this basis we search for long-term variations in sea level heights measured in the tidal stations of Catania, Messina and Marseille, and associate them with the concomitant phase of dominant modes of interannual-to-decadal climate variability in the Euro-Mediterranean sector. We focus on the atmospheric North Atlantic Oscillation (NAO) and on the Adriatic-Ionian Bimodal Oscillating System (BiOS) to illustrate the grand variability in sea level teleconnections during the last four decades. In particular, observations reveal a strong imprint of both NAO and BiOS on all sea level records in the 21st century, when NAO and BiOS are inversely correlated. In the 1990s, a well known period of persistent positive NAO anomalies, the NAO imprint on sea level becomes weaker compared to the most recent period, although it remains clear on decadal trends, while the BiOS shows very weak positive variability. In the 1970s and early 1980s, when the NAO was on a neutral phase with weak variability, the NAO imprint on sea levels is weakest, and sea levels in Marseille and Sicily anticorrelate with each other, in contrast to the positive correlations found during the later periods. Based on these observational evidence, we discuss how our modeling results provide a basis to understand the local dynamics that contributed to determine such observed decadal variability.

  15. Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates

    PubMed Central

    Romanchuk, Artur; Chang, Jeff H.; Mukhtar, M. Shahid; Cherkis, Karen; Roach, Jeff; Grant, Sarah R.; Jones, Corbin D.; Dangl, Jeffery L.

    2011-01-01

    Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae, we screened 11 diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan-genome of the species. PMID:21799664

  16. Quantum Dynamics Simulations Reveal Vibronic Effects on the Optical Properties of [n]Cycloparaphenylenes.

    PubMed

    Reddy, V Sivaranjana; Camacho, Cristopher; Xia, Jianlong; Jasti, Ramesh; Irle, Stephan

    2014-09-09

    The size-dependent ultraviolet/visible photophysical property trends of [n]cycloparaphenylenes ([n]CPPs, n = 6, 8, and 10) are theoretically investigated using quantum dynamics simulations. For geometry optimizations on the ground- and excited-state Born-Oppenheimer potential energy surfaces (PESs), we employ density functional theory (DFT) and time-dependent DFT calculations. Harmonic normal-mode analyses are carried out for the electronic ground state at Franck-Condon geometries. A diabatic Hamiltonian, comprising four low-lying singlet excited electronic states and 26 vibrational degrees of freedom of CPP, is constructed within the linear vibronic coupling (VC) model to elucidate the absorption spectral features in the range of 300-500 nm. Quantum nuclear dynamics is simulated within the multiconfiguration time-dependent Hartree approach to calculate the vibronic structure of the excited electronic states. The symmetry-forbidden S0 → S1 transition appears in the longer wavelength region of the spectrum with weak intensity due to VC. It is found that the Jahn-Teller and pseudo-Jahn-Teller effects in the doubly degenerate S2 and S3 electronic states are essential in the quantitative interpretation of the experimental observation of a broad absorption peak around 340 nm. The vibronic mixing of the S1 state with higher electronic states is responsible for the efficient photoluminescence from the S1 state. The fluorescence properties are characterized on the basis of the stationary points of the excited-state PESs. The findings reveal that vibronic effects become important in determining the photophysical properties of CPPs with increased ring size.

  17. Clonal expansion during Staphylococcus aureus infection dynamics reveals the effect of antibiotic intervention.

    PubMed

    McVicker, Gareth; Prajsnar, Tomasz K; Williams, Alexander; Wagner, Nelly L; Boots, Michael; Renshaw, Stephen A; Foster, Simon J

    2014-02-01

    To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population "bottleneck" whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where

  18. Dynamic auxin transport patterns preceding vein formation revealed by live-imaging of Arabidopsis leaf primordia

    PubMed Central

    Marcos, Danielle; Berleth, Thomas

    2014-01-01

    Self-regulatory patterning mechanisms capable of generating biologically meaningful, yet unpredictable cellular patterns offer unique opportunities for obtaining mathematical descriptions of underlying patterning systems properties. The networks of higher-order veins in leaf primordia constitute such a self-regulatory system. During the formation of higher-order veins, vascular precursors are selected from a homogenous field of subepidermal cells in unpredictable positions to eventually connect in complex cellular networks. Auxin transport routes have been implicated in this selection process, but understanding of their role in vascular patterning has been limited by our inability to monitor early auxin transport dynamics in vivo. Here we describe a live-imaging system in emerging Arabidopsis thaliana leaves that uses a PIN1:GFP reporter to visualize auxin transport routes and an Athb8:YFP reporter as a marker for vascular commitment. Live-imaging revealed common features initiating the formation of all higher-order veins. The formation of broad PIN1 expression domains is followed by their restriction, leading to sustained, elevated PIN1 expression in incipient procambial cells files, which then express Athb8. Higher-order PIN1 expression domains (hPEDs) are initiated as freely ending domains that extend toward each other and sometimes fuse with them, creating connected domains. During the restriction and specification phase, cells in wider hPEDs are partitioned into vascular and non-vascular fates: Central cells acquire a coordinated cell axis and express elevated PIN1 levels as well as the pre-procambial marker Athb8, while edge cells downregulate PIN1 and remain isodiametric. The dynamic nature of the early selection process is underscored by the instability of early hPEDs, which can result in dramatic changes in vascular network architecture prior to Athb8 expression, which is correlated with the promotion onto vascular cell fate. PMID:24966861

  19. Electrocorticography reveals the temporal dynamics of posterior parietal cortical activity during recognition memory decisions

    PubMed Central

    Gonzalez, Alex; Hutchinson, J. Benjamin; Uncapher, Melina R.; Chen, Janice; LaRocque, Karen F.; Foster, Brett L.; Rangarajan, Vinitha; Parvizi, Josef; Wagner, Anthony D.

    2015-01-01

    Theories of the neurobiology of episodic memory predominantly focus on the contributions of medial temporal lobe structures, based on extensive lesion, electrophysiological, and imaging evidence. Against this backdrop, functional neuroimaging data have unexpectedly implicated left posterior parietal cortex (PPC) in episodic retrieval, revealing distinct activation patterns in PPC subregions as humans make memory-related decisions. To date, theorizing about the functional contributions of PPC has been hampered by the absence of information about the temporal dynamics of PPC activity as retrieval unfolds. Here, we leveraged electrocorticography to examine the temporal profile of high gamma power (HGP) in dorsal PPC subregions as participants made old/new recognition memory decisions. A double dissociation in memory-related HGP was observed, with activity in left intraparietal sulcus (IPS) and left superior parietal lobule (SPL) differing in time and sign for recognized old items (Hits) and correctly rejected novel items (CRs). Specifically, HGP in left IPS increased for Hits 300–700 ms poststimulus onset, and decayed to baseline ∼200 ms preresponse. By contrast, HGP in left SPL increased for CRs early after stimulus onset (200−300 ms) and late in the memory decision (from 700 ms to response). These memory-related effects were unique to left PPC, as they were not observed in right PPC. Finally, memory-related HGP in left IPS and SPL was sufficiently reliable to enable brain-based decoding of the participant’s memory state at the single-trial level, using multivariate pattern classification. Collectively, these data provide insights into left PPC temporal dynamics as humans make recognition memory decisions. PMID:26283375

  20. Particle Motion Analysis Reveals Nanoscale Bond Characteristics and Enhances Dynamic Range for Biosensing.

    PubMed

    Visser, Emiel W A; van IJzendoorn, Leo J; Prins, Menno W J

    2016-03-22

    Biofunctionalized colloidal particles are widely used as labels in bioanalytical assays, lab-on-chip devices, biophysical research, and in studies on live biological systems. With detection resolution going down to the level of single particles and single molecules, understanding the nature of the interaction of the particles with surfaces and substrates becomes of paramount importance. Here, we present a comprehensive study of motion patterns of colloidal particles maintained in close proximity to a substrate by short molecular tethers (40 nm). The motion of the particles (500-1000 nm) was optically tracked with a very high localization accuracy (below 3 nm). A surprisingly large variation in motion patterns was observed, which can be attributed to properties of the particle-molecule-substrate system, namely the bond number, the nature of the bond, particle protrusions, and substrate nonuniformities. Experimentally observed motion patterns were compared to numerical Monte Carlo simulations, revealing a close correspondence between the observed motion patterns and properties of the molecular system. Particles bound via single tethers show distinct disc-, ring-, and bell-shaped motion patterns, where the ring- and bell-shaped patterns are caused by protrusions on the particle in the direct vicinity of the molecular attachment point. Double and triple tethered particles exhibit stripe-shaped and triangular-shaped motion patterns, respectively. The developed motion pattern analysis allows for discrimination between particles bound by different bond types, which opens the possibility to improve the limit of detection and the dynamic range of bioanalytical assays, with a projected increase of dynamic range by nearly 2 orders of magnitude.

  1. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  2. Trends in genome dynamics among major orders of insects revealed through variations in protein families.

    PubMed

    Rappoport, Nadav; Linial, Michal

    2015-08-07

    Insects belong to a class that accounts for the majority of animals on earth. With over one million identified species, insects display a huge diversity and occupy extreme environments. At present, there are dozens of fully sequenced insect genomes that cover a range of habitats, social behavior and morphologies. In view of such diverse collection of genomes, revealing evolutionary trends and charting functional relationships of proteins remain challenging. We analyzed the relatedness of 17 complete proteomes representative of proteomes from insects including louse, bee, beetle, ants, flies and mosquitoes, as well as an out-group from the crustaceans. The analyzed proteomes mostly represented the orders of Hymenoptera and Diptera. The 287,405 protein sequences from the 18 proteomes were automatically clustered into 20,933 families, including 799 singletons. A comprehensive analysis based on statistical considerations identified the families that were significantly expanded or reduced in any of the studied organisms. Among all the tested species, ants are characterized by an exceptionally high rate of family gain and loss. By assigning annotations to hundreds of species-specific families, the functional diversity among species and between the major clades (Diptera and Hymenoptera) is revealed. We found that many species-specific families are associated with receptor signaling, stress-related functions and proteases. The highest variability among insects associates with the function of transposition and nucleic acids processes (collectively coined TNAP). Specifically, the wasp and ants have an order of magnitude more TNAP families and proteins relative to species that belong to Diptera (mosquitoes and flies). An unsupervised clustering methodology combined with a comparative functional analysis unveiled proteomic signatures in the major clades of winged insects. We propose that the expansion of TNAP families in Hymenoptera potentially contributes to the accelerated

  3. Phosphoproteome analysis of B. cinerea in response to different plant-based elicitors.

    PubMed

    Liñeiro, Eva; Chiva, Cristina; Cantoral, Jesús M; Sabido, Eduard; Fernández-Acero, Francisco Javier

    2016-04-29

    The phytopathogen Botrytis cinerea is a ubiquitous fungus with a high capacity to adapt its metabolism to different hosts and environmental conditions in order to deploy a variety of virulence and pathogenicity factors and develop a successful plant infection. Here we report the first comparative phosphoproteomic study of B. cinerea, aimed to analyze the phosphoprotein composition of the fungus and its changes under different phenotypical conditions induced by two different carbon sources as plant based elicitors: glucose and deproteinized tomato cell wall (TCW). A total of 2854 and 2269 different phosphosites (2883 and 1137 phosphopeptides) were identified in glucose and TCW respectively, which map to 1338 phosphoproteins in glucose and 733 in TCW. Out of the identified phosphoproteins, 173 were exclusively found when glucose was the only carbon source and 11 when the carbon source was TCW. Differences in the pattern of phosphorylation-sites were also detected according to the carbon source. Gene ontology classification of the identified phosphoproteins showed that most of the characteristic proteins of the different carbon sources were related to signalling and transmembrane transport, thus highlighting the importance of these processes in the fungal adaptation to the surrounding conditions. The characterization of the B. cinerea phosphoproteome under different induction conditions reported here is the first comparative phosphoproteomic approach in this model phytopathogenic fungus. The identified phosphopeptides contribute to expand the map of known phosphoproteins in this pathogen and the observed changes according to the used carbon source contribute to understand the adaptation of the fungus to the environment changes. This knowledge improves the understanding of the adaptation mechanism, defines the role of the phosphoproteins involved in this process, and enables the advance in the design of novel strategies against the fungi. Copyright © 2016 Elsevier B

  4. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    PubMed

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  5. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms

    PubMed Central

    Adams, Nancy E.; Maurelli, Anthony T.

    2015-01-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB–RB transitions. PMID:25998263

  6. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress.

    PubMed

    Hu, Xiuli; Wu, Liuji; Zhao, Feiyun; Zhang, Dayong; Li, Nana; Zhu, Guohui; Li, Chaohao; Wang, Wei

    2015-01-01

    Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.

  7. Characterization of 4-HNE Modified L-FABP Reveals Alterations in Structural and Functional Dynamics

    PubMed Central

    Smathers, Rebecca L.; Fritz, Kristofer S.; Galligan, James J.; Shearn, Colin T.; Reigan, Philip; Marks, Michael J.; Petersen, Dennis R.

    2012-01-01

    4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd1 = 0.395 µM and Kd2 = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a

  8. Intravital Placenta Imaging Reveals Microcirculatory Dynamics Impact on Sequestration and Phagocytosis of Plasmodium-Infected Erythrocytes

    PubMed Central

    de Moraes, Luciana Vieira; Tadokoro, Carlos Eduardo; Gómez-Conde, Iván; Olivieri, David N.; Penha-Gonçalves, Carlos

    2013-01-01

    Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE. PMID:23382682

  9. Dynamic transcriptome changes during adipose tissue energy expenditure reveal critical roles for long noncoding RNA regulators

    PubMed Central

    Yoon, Myeong Jin; Kim, Hye-Jin; LO, Kinyui Alice; Zhang, Zhi-chun; Xu, Dan; Siang, Diana Teh Chee; Walet, Arcinas Camille Esther; Xu, Shao-hai; Chia, Sook-Yoong; Chen, Peng; Yang, Hongyuan; Ghosh, Sujoy

    2017-01-01

    Enhancing brown fat activity and promoting white fat browning are attractive therapeutic strategies for treating obesity and associated metabolic disorders. To provide a comprehensive picture of the gene regulatory network in these processes, we conducted a series of transcriptome studies by RNA sequencing (RNA-seq) and quantified the mRNA and long noncoding RNA (lncRNA) changes during white fat browning (chronic cold exposure, beta-adrenergic agonist treatment, and intense exercise) and brown fat activation or inactivation (acute cold exposure or thermoneutrality, respectively). mRNA–lncRNA coexpression networks revealed dynamically regulated lncRNAs to be largely embedded in nutrient and energy metabolism pathways. We identified a brown adipose tissue–enriched lncRNA, lncBATE10, that was governed by the cAMP-cAMP response element-binding protein (Creb) axis and required for a full brown fat differentiation and white fat browning program. Mechanistically, lncBATE10 can decoy Celf1 from Pgc1α, thereby protecting Pgc1α mRNA from repression by Celf1. Together, these studies provide a comprehensive data framework to interrogate the transcriptomic changes accompanying energy homeostasis transition in adipose tissue. PMID:28763438

  10. EEG source reconstruction reveals frontal-parietal dynamics of spatial conflict processing.

    PubMed

    Cohen, Michael X; Ridderinkhof, K Richard

    2013-01-01

    Cognitive control requires the suppression of distracting information in order to focus on task-relevant information. We applied EEG source reconstruction via time-frequency linear constrained minimum variance beamforming to help elucidate the neural mechanisms involved in spatial conflict processing. Human subjects performed a Simon task, in which conflict was induced by incongruence between spatial location and response hand. We found an early (∼200 ms post-stimulus) conflict modulation in stimulus-contralateral parietal gamma (30-50 Hz), followed by a later alpha-band (8-12 Hz) conflict modulation, suggesting an early detection of spatial conflict and inhibition of spatial location processing. Inter-regional connectivity analyses assessed via cross-frequency coupling of theta (4-8 Hz), alpha, and gamma power revealed conflict-induced shifts in cortical network interactions: Congruent trials (relative to incongruent trials) had stronger coupling between frontal theta and stimulus-contrahemifield parietal alpha/gamma power, whereas incongruent trials had increased theta coupling between medial frontal and lateral frontal regions. These findings shed new light into the large-scale network dynamics of spatial conflict processing, and how those networks are shaped by oscillatory interactions.

  11. Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modeling.

    PubMed

    Correnti, Jason M; Cook, Daniel; Aksamitiene, Edita; Swarup, Aditi; Ogunnaike, Babatunde; Vadigepalli, Rajanikanth; Hoek, Jan B

    2014-11-10

    Following partial hepatectomy, the liver initiates a regenerative program involving hepatocyte priming and replication driven by coordinated cytokine and growth factor actions. We investigated the mechanisms underlying Adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. Adn-/- mice showed delayed onset of hepatocyte replication, but accelerated cell cycle progression relative to wild-type mice, suggesting Adn has multiple effects fine-tuning the kinetics of liver regeneration. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn-/- mice. We employed this computational model to evaluate the underlying regulatory mechanisms. Our analysis predicted that Adn is required for an efficient early cytokine response to partial hepatectomy, but is inhibitory to later growth factor actions. Consistent with this prediction, Adn knockout reduced hepatocyte responses to IL-6 during the priming phase, but enhanced growth factor levels through peak hepatocyte replication. By contrast, supraphysiological concentrations of Adn resulting from rosiglitazone treatment suppressed regeneration by reducing growth factor levels during S phase, consistent with computational predictions. Together, these results revealed that Adn fine-tunes the progression of liver regeneration through dynamically modulating molecular mediator networks and cellular interactions within the liver. This article is protected by copyright. All rights reserved.

  12. Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modelling.

    PubMed

    Correnti, Jason M; Cook, Daniel; Aksamitiene, Edita; Swarup, Aditi; Ogunnaike, Babatunde; Vadigepalli, Rajanikanth; Hoek, Jan B

    2015-01-15

    Following partial hepatectomy, the liver initiates a regenerative programme involving hepatocyte priming and replication driven by the coordinated actions of cytokine and growth factors. We investigated the mechanisms underlying adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. Adn(-/-) mice showed delayed onset of hepatocyte replication, but accelerated cell cycle progression relative to wild-type mice, suggesting Adn has multiple effects fine-tuning the kinetics of liver regeneration. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn(-/-) mice. We employed this computational model to evaluate the underlying regulatory mechanisms. Our analysis predicted that Adn is required for an efficient early cytokine response to partial hepatectomy, but is inhibitory to later growth factor actions. Consistent with this prediction, Adn knockout reduced hepatocyte responses to interleukin-6 during the priming phase, but enhanced growth factor levels through peak hepatocyte replication. By contrast, supraphysiological concentrations of Adn resulting from rosiglitazone treatment suppressed regeneration by reducing growth factor levels during S phase, consistent with computational predictions. Together, these results revealed that Adn fine-tunes the progression of liver regeneration through dynamically modulating molecular mediator networks and cellular interactions within the liver.

  13. Diversity of sharp-wave–ripple LFP signatures reveals differentiated brain-wide dynamical events

    PubMed Central

    Ramirez-Villegas, Juan F.; Logothetis, Nikos K.; Besserve, Michel

    2015-01-01

    Sharp-wave–ripple (SPW-R) complexes are believed to mediate memory reactivation, transfer, and consolidation. However, their underlying neuronal dynamics at multiple scales remains poorly understood. Using concurrent hippocampal local field potential (LFP) recordings and functional MRI (fMRI), we study local changes in neuronal activity during SPW-R episodes and their brain-wide correlates. Analysis of the temporal alignment between SPW and ripple components reveals well-differentiated SPW-R subtypes in the CA1 LFP. SPW-R–triggered fMRI maps show that ripples aligned to the positive peak of their SPWs have enhanced neocortical metabolic up-regulation. In contrast, ripples occurring at the trough of their SPWs relate to weaker neocortical up-regulation and absent subcortical down-regulation, indicating differentiated involvement of neuromodulatory pathways in the ripple phenomenon mediated by long-range interactions. To our knowledge, this study provides the first evidence for the existence of SPW-R subtypes with differentiated CA1 activity and metabolic correlates in related brain areas, possibly serving different memory functions. PMID:26540729

  14. Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

    NASA Astrophysics Data System (ADS)

    Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

    2016-04-01

    Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

  15. Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory.

    PubMed

    Qu, Wenchao; Robert, Christelle A M; Erb, Matthias; Hibbard, Bruce E; Paven, Maxim; Gleede, Tassilo; Riehl, Barbara; Kersting, Lena; Cankaya, Aylin S; Kunert, Anna T; Xu, Youwen; Schueller, Michael J; Shea, Colleen; Alexoff, David; Lee, So Jeong; Fowler, Joanna S; Ferrieri, Richard A

    2016-10-01

    The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a major pest of maize (Zea mays) that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using (11)C with positron emission tomography, root autoradiography, and radiometabolite flux analysis to understand maize tolerance to WCR Our results reveal that WCR attack induces specific patterns of lateral root growth that are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of Gln. Finally, the regrowth zones of WCR-attacked roots show an increase in Gln turnover, which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin biosynthesis flux network as a promising marker for induced WCR tolerance.

  16. Host–pathogen evolutionary signatures reveal dynamics and future invasions of vampire bat rabies

    PubMed Central

    Streicker, Daniel G.; Winternitz, Jamie C.; Satterfield, Dara A.; Condori-Condori, Rene Edgar; Broos, Alice; Tello, Carlos; Recuenco, Sergio; Velasco-Villa, Andrés; Altizer, Sonia; Valderrama, William

    2016-01-01

    Anticipating how epidemics will spread across landscapes requires understanding host dispersal events that are notoriously difficult to measure. Here, we contrast host and virus genetic signatures to resolve the spatiotemporal dynamics underlying geographic expansions of vampire bat rabies virus (VBRV) in Peru. Phylogenetic analysis revealed recent viral spread between populations that, according to extreme geographic structure in maternally inherited host mitochondrial DNA, appeared completely isolated. In contrast, greater population connectivity in biparentally inherited nuclear microsatellites explained the historical limits of invasions, suggesting that dispersing male bats spread VBRV between genetically isolated female populations. Host nuclear DNA further indicated unanticipated gene flow through the Andes mountains connecting the VBRV-free Pacific coast to the VBRV-endemic Amazon rainforest. By combining Bayesian phylogeography with landscape resistance models, we projected invasion routes through northern Peru that were validated by real-time livestock rabies mortality data. The first outbreaks of VBRV on the Pacific coast of South America could occur by June 2020, which would have serious implications for agriculture, wildlife conservation, and human health. Our results show that combining host and pathogen genetic data can identify sex biases in pathogen spatial spread, which may be a widespread but underappreciated phenomenon, and demonstrate that genetic forecasting can aid preparedness for impending viral invasions. PMID:27621441

  17. Host-pathogen evolutionary signatures reveal dynamics and future invasions of vampire bat rabies.

    PubMed

    Streicker, Daniel G; Winternitz, Jamie C; Satterfield, Dara A; Condori-Condori, Rene Edgar; Broos, Alice; Tello, Carlos; Recuenco, Sergio; Velasco-Villa, Andrés; Altizer, Sonia; Valderrama, William

    2016-09-27

    Anticipating how epidemics will spread across landscapes requires understanding host dispersal events that are notoriously difficult to measure. Here, we contrast host and virus genetic signatures to resolve the spatiotemporal dynamics underlying geographic expansions of vampire bat rabies virus (VBRV) in Peru. Phylogenetic analysis revealed recent viral spread between populations that, according to extreme geographic structure in maternally inherited host mitochondrial DNA, appeared completely isolated. In contrast, greater population connectivity in biparentally inherited nuclear microsatellites explained the historical limits of invasions, suggesting that dispersing male bats spread VBRV between genetically isolated female populations. Host nuclear DNA further indicated unanticipated gene flow through the Andes mountains connecting the VBRV-free Pacific coast to the VBRV-endemic Amazon rainforest. By combining Bayesian phylogeography with landscape resistance models, we projected invasion routes through northern Peru that were validated by real-time livestock rabies mortality data. The first outbreaks of VBRV on the Pacific coast of South America could occur by June 2020, which would have serious implications for agriculture, wildlife conservation, and human health. Our results show that combining host and pathogen genetic data can identify sex biases in pathogen spatial spread, which may be a widespread but underappreciated phenomenon, and demonstrate that genetic forecasting can aid preparedness for impending viral invasions.

  18. Spectral properties and dynamics of gold nanorods revealed by EMCCD-based spectral phasor method.

    PubMed

    Chen, Hongtao; Gratton, Enrico; Digman, Michelle A

    2015-04-01

    Gold nanorods (NRs) with tunable plasmon-resonant absorption in the near-infrared region have considerable advantages over organic fluorophores as imaging agents due to their brightness and lack of photobleaching. However, the luminescence spectral properties of NRs have not been fully characterized at the single particle level due to lack of proper analytic tools. Here, we present a spectral phasor analysis method that allows investigations of NRs' spectra at single particle level showing the spectral variance and providing spatial information during imaging. The broad phasor distribution obtained by the spectral phasor analysis indicates that spectra of NRs are different from particle to particle. NRs with different spectra can be identified in images with high spectral resolution. The spectral behaviors of NRs under different imaging conditions, for example, different excitation powers and wavelengths, were revealed by our laser-scanning multiphoton microscope using a high-resolution spectrograph with imaging capability. Our results prove that the spectral phasor method is an easy and efficient tool in hyper-spectral imaging analysis to unravel subtle changes of the emission spectrum. We applied this method to study the spectral dynamics of NRs during direct optical trapping and by optothermal trapping. Interestingly, different spectral shifts were observed in both trapping phenomena.

  19. Revealing spatio-spectral electroencephalographic dynamics of musical mode and tempo perception by independent component analysis

    PubMed Central

    2014-01-01

    Background Music conveys emotion by manipulating musical structures, particularly musical mode- and tempo-impact. The neural correlates of musical mode and tempo perception revealed by electroencephalography (EEG) have not been adequately addressed in the literature. Method This study used independent component analysis (ICA) to systematically assess spatio-spectral EEG dynamics associated with the changes of musical mode and tempo. Results Empirical results showed that music with major mode augmented delta-band activity over the right sensorimotor cortex, suppressed theta activity over the superior parietal cortex, and moderately suppressed beta activity over the medial frontal cortex, compared to minor-mode music, whereas fast-tempo music engaged significant alpha suppression over the right sensorimotor cortex. Conclusion The resultant EEG brain sources were comparable with previous studies obtained by other neuroimaging modalities, such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET). In conjunction with advanced dry and mobile EEG technology, the EEG results might facilitate the translation from laboratory-oriented research to real-life applications for music therapy, training and entertainment in naturalistic environments. PMID:24581119

  20. Molecular dynamics simulation revealed binding of nucleotide inhibitors to ZIKV polymerase over 444 nanoseconds.

    PubMed

    Elfiky, Abdo A; Elshemey, Wael M

    2017-09-18

    In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2' carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti-hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide-range antiviral drug. © 2017 Wiley Periodicals, Inc.