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Chia-Lang Hsu, Jian-Kai Wang, Pei-Chun Lu, Hsuan-Cheng Huang, Hsueh-Fen Juan
Summary: Large-scale phosphoproteomics studies have improved our understanding of dynamic cellular signaling, but the downstream analysis of phosphoproteomics data is still a bottleneck. We develop DynaPho, a useful web-based tool providing comprehensive and in-depth analyses of time-course phosphoproteomics data, making analysis intuitive and accessible to non-bioinformatics experts. The tool currently implements five analytic modules, which reveal the transition of biological pathways, kinase activity, dynamics of interaction networks and the predicted kinase-substrate associations...
July 7, 2017: Bioinformatics
Deane F Mosher, Emily M Wilkerson, Keren B Turton, Alexander S Hebert, Joshua J Coon
We recently identified and quantified >7,000 proteins in non-activated human peripheral blood eosinophils using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and described phosphoproteomic changes that accompany acute activation of eosinophils by interleukin-5 (IL5) (1). These data comprise a treasure trove of information about eosinophils. We illustrate the power of label-free LC-MS/MS quantification by considering four examples: complexity of eosinophil STATs, contribution of immunoproteasome subunits to eosinophil proteasomes, complement of integrin subunits, and contribution of platelet proteins originating from platelet-eosinophil complexes to the overall proteome...
2017: Frontiers in Medicine
Yonggen Jia, Jean-Baptiste Marq, Hugo Bisio, Damien Jacot, Christina Mueller, Lu Yu, Jyoti Choudhary, Mathieu Brochet, Dominique Soldati-Favre
Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis...
October 13, 2017: EMBO Journal
Xiaoyue Jiang, Ryan Bomgarden, Joseph Brown, Devin L Drew, Aaron M Robitaille, Rosa Viner, Andreas R Huhmer
Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMT) have become a powerful platform for simultaneous, large scale phospho-proteome site identification and quantitation. In order to improve the accuracy of isobaric tag-based quantitation in complex proteomic samples, MS3-based acquisition methods such as Synchronous Precursor Selection (SPS) have been used. However, the method suffers from lower peptide identification rates when applied to enriched phosphopeptide samples compared to unmodified samples due to differences in phosphopeptide fragmentation patterns during tandem MS...
October 12, 2017: Journal of Proteome Research
Stephanie Munk, Jón Otti Sigurðsson, Zhenyu Xiao, Tanveer Singh Batth, Giulia Franciosa, Louise von Stechow, Andres Joaquin Lopez-Contreras, Alfred Cornelis Otto Vertegaal, Jesper Velgaard Olsen
The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites...
October 10, 2017: Cell Reports
Taras Stasyk, Lukas Alfons Huber
Here, we describe the detailed step-by-step protocol for detection of phosphoproteins in two-dimensional difference gel electrophoresis (DIGE) gels. A standard DIGE protocol is combined with subsequent post-staining with phosphospecific fluorescent dye. The combination of these two methods complements DIGE-based proteome profiling by fluorescence detection of phosphoproteins in the same gel providing additional possibility for sensitive and accurate quantification of the differentially regulated phosphoproteins in biological samples...
2018: Methods in Molecular Biology
Qingke Duan, Hengqiang Zhao, Zhengle Zhang, Hehe Li, Heshui Wu, Qiang Shen, Chunyou Wang, Tao Yin
One of the main causations of the poor prognosis of pancreatic cancer is the lack of effective chemotherapies. Gemcitabine is a widely used chemotherapeutic drug, but limited therapeutic efficacy is achieved due to chemoresistance. Recent studies demonstrated that the presence of cancer stem cells may lead to the failure of chemotherapy. Moreover, gemcitabine can promote the stemness of pancreatic cancer cells. We detected the alterations in protein phosphorylation and signaling pathways in pancreatic cancer cells after gemcitabine treatment using iTRAQ labeling LC-MS/MS, because it was featured with the advantages of strong separation ability and analysis range...
October 10, 2017: Scientific Reports
Rubin N Joshi, Nadine A Binai, Francesco Marabita, Zhenhua Sui, Amnon Altman, Albert J R Heck, Jesper Tegnér, Angelika Schmidt
Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4(+)CD25(-) T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively...
2017: Frontiers in Immunology
Qian-Ru Li, Xiu-Min Yan, Lin Guo, Jia Li, Yi Zang
AMP-activated protein kinase (AMPK) is an energy sensor that couples the cellular energy state with basic biological processes. AMPK is thought to be linked with cell division although the underlying mechanisms remain largely unknown. Here, we show that AMPK functionally participates throughout cell division and that AMPK catalytic subunits, especially α2, are sequentially associated with separate mitotic apparatus. Using quantitative phosphoproteomics analysis, we found that the strong direct substrate KIF4A is phosphorylated by AMPK at Ser801...
September 14, 2017: Journal of Molecular Cell Biology
Brent M Kuenzi, Lily L Remsing Rix, Paul A Stewart, Bin Fang, Fumi Kinose, Annamarie T Bryant, Theresa A Boyle, John M Koomen, Eric B Haura, Uwe Rix
Targeted drugs are effective when they directly inhibit strong disease drivers, but only a small fraction of diseases feature defined actionable drivers. Alternatively, network-based approaches can uncover new therapeutic opportunities. Applying an integrated phenotypic screening, chemical and phosphoproteomics strategy, here we describe the anaplastic lymphoma kinase (ALK) inhibitor ceritinib as having activity across several ALK-negative lung cancer cell lines and identify new targets and network-wide signaling effects...
October 9, 2017: Nature Chemical Biology
Matthew Y Lim, Jonathon O'Brien, Joao A Paulo, Steven P Gygi
Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context(1). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale(2). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry...
October 6, 2017: Journal of Proteome Research
Cheng-Kang Chiang, Aleksander Tworak, Brian M Kevany, Bo Xu, Janice Mayne, Zhibin Ning, Daniel Figeys, Krzysztof Palczewski
One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration...
October 4, 2017: Journal of Biological Chemistry
Mengfang Du, Xiaoguang Liu, Nana Ma, Xiaoming Liu, Jizhen Wei, Xinming Yin, Shutang Zhou, Rafaeli Ada, Qisheng Song, Shiheng An
Chemical signaling plays a critical role in the behavior and physiology of many animals. Female insects, as a number of many other animals, release sex pheromones to attract males for mating. The evolutionary and ecological success of insects therefore hinges on their ability to precisely mediate (including initiation and termination) pheromone biosynthesis. Pheromone biosynthesis activating neuropeptide (PBAN) acts directly on pheromone glands to regulate sex pheromone production using Ca(2+) and cyclic-AMP as secondary messengers in the majority of species...
October 4, 2017: Molecular & Cellular Proteomics: MCP
Kiyoshi Isobe, Hyun Jun Jung, Chin-Rang Yang, J'Neka Claxton, Pablo Sandoval, Maurice B Burg, Viswanathan Raghuram, Mark A Knepper
G protein stimulatory α-subunit (Gαs)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the Gαs-coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells...
October 2, 2017: Proceedings of the National Academy of Sciences of the United States of America
Loreta Bllaci, Silje Bøen Torsetnes, Celina Katarzyna Wierzbicka, Sudhirkumar Shinde, Börje Sellergren, Adelina Rogowska-Wrzesinska, Ole Nørregaard Jensen
Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells...
October 3, 2017: Analytical Chemistry
Sohrab Saraei, Tomi Suomi, Otto Kauko, Laura L Elo
Motivation: Global centering-based normalization is a commonly-used normalization approach in mass spectrometry-based label-free proteomics. It scales the peptide abundances to have the same median intensities, based on an assumption that the majority of abundances remain the same across the samples. However, especially in phosphoproteomics, this assumption can introduce bias, as the samples are enriched during sample preparation which can mask the underlying biological changes. To address this possible bias, phosphopeptides quantified in both enriched and non-enriched samples can be used to calculate factors that mitigate the bias...
September 15, 2017: Bioinformatics
Danica D Wiredja, Marzieh Ayati, Sahar Mazhar, Jaya Sangodkar, Sean Maxwell, Daniela Schlatzer, Goutham Narla, Mehmet Koyutürk, Mark R Chance
Activation of protein phosphatase 2A (PP2A) is a promising anti-cancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel Small Molecule Activator of PP2A (SMAP)...
September 29, 2017: Proteomics
Westa Domanova, James R Krycer, Rima Chaudhuri, Pengyi Yang, Fatemeh Vafaee, Daniel J Fazakerley, Sean J Humphrey, David E James, Zdenka Kuncic
[This corrects the article DOI: 10.1371/journal.pone.0157763.].
2017: PloS One
Scott M Thompson, Danielle E Jondal, Kim A Butters, Bruce E Knudsen, Jill L Anderson, Matthew P Stokes, Xiaoying Jia, Joseph P Grande, Lewis R Roberts, Matthew R Callstrom, David A Woodrum
PURPOSE: The aims of the present study were two-fold: first, to test the hypothesis that heat stress induces MET and EGFR signaling in hepatocellular carcinoma (HCC) cells and inhibition of this signaling decreases HCC clonogenic survival; and second, to identify signaling pathways associated with heat-stress induced MET signaling. MATERIALS AND METHODS: MET(+) and EGFR(+) HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry...
September 27, 2017: International Journal of Hyperthermia
Sharon K Kuss-Duerkop, Juan Wang, Ignacio Mena, Kris White, Giorgi Metreveli, Ramanavelan Sakthivel, Miguel A Mata, Raquel Muñoz-Moreno, Xiang Chen, Florian Krammer, Michael S Diamond, Zhijian J Chen, Adolfo García-Sastre, Beatriz M A Fontoura
Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection...
September 2017: PLoS Pathogens
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