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Sample records for cells integral proteins

  1. The Unfolded Protein Response Is Induced by the Cell Wall Integrity Mitogen-activated Protein Kinase Signaling Cascade and Is Required for Cell Wall Integrity in Saccharomyces cerevisiae

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    Scrimale, Thomas; Didone, Louis; de Mesy Bentley, Karen L.; Krysan, Damian J.

    2009-01-01

    The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expres...

  2. High yield cell-free production of integral membrane proteins without refolding or detergents.

    Science.gov (United States)

    Wuu, Jessica J; Swartz, James R

    2008-05-01

    Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

  3. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

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    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  4. Protein-Mediated Interactions of Pancreatic Islet Cells

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    Paolo Meda

    2013-01-01

    Full Text Available The islets of Langerhans collectively form the endocrine pancreas, the organ that is soley responsible for insulin secretion in mammals, and which plays a prominent role in the control of circulating glucose and metabolism. Normal function of these islets implies the coordination of different types of endocrine cells, noticeably of the beta cells which produce insulin. Given that an appropriate secretion of this hormone is vital to the organism, a number of mechanisms have been selected during evolution, which now converge to coordinate beta cell functions. Among these, several mechanisms depend on different families of integral membrane proteins, which ensure direct (cadherins, N-CAM, occludin, and claudins and paracrine communications (pannexins between beta cells, and between these cells and the other islet cell types. Also, other proteins (integrins provide communication of the different islet cell types with the materials that form the islet basal laminae and extracellular matrix. Here, we review what is known about these proteins and their signaling in pancreatic β-cells, with particular emphasis on the signaling provided by Cx36, given that this is the integral membrane protein involved in cell-to-cell communication, which has so far been mostly investigated for effects on beta cell functions.

  5. Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

    Science.gov (United States)

    Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

    2016-11-01

    Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

  6. The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

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    Ashley R Long

    Full Text Available The ADP/ATP Carrier (AAC is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

  7. The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

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    Miguel-Rojas, Cristina; Hera, Concepcion

    2016-01-01

    F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  8. Recombinant protein production from stable mammalian cell lines and pools.

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    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    International Nuclear Information System (INIS)

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-01-01

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [ 3 H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [ 3 H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-κB, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  10. Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

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    Guruceaga, Elizabeth; Garin-Muga, Alba; Prieto, Gorka; Bejarano, Bartolomé; Marcilla, Miguel; Marín-Vicente, Consuelo; Perez-Riverol, Yasset; Casal, J Ignacio; Vizcaíno, Juan Antonio; Corrales, Fernando J; Segura, Victor

    2017-12-01

    The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

  11. Integrating cell biology and proteomic approaches in plants.

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    Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

    2017-10-03

    Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of

  12. Functional dynamics of cell surface membrane proteins.

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    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    International Nuclear Information System (INIS)

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-01-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [ 35 S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [ 3 H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125 I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [ 3 H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

  14. Live-cell and super-resolution imaging reveal that the distribution of wall-associated protein A is correlated with the cell chain integrity of Streptococcus mutans.

    Science.gov (United States)

    Li, Y; Liu, Z; Zhang, Y; Su, Q P; Xue, B; Shao, S; Zhu, Y; Xu, X; Wei, S; Sun, Y

    2015-10-01

    Streptococcus mutans is a primary pathogen responsible for dental caries. It has an outstanding ability to form biofilm, which is vital for virulence. Previous studies have shown that knockout of Wall-associated protein A (WapA) affects cell chain and biofilm formation of S. mutans. As a surface protein, the distribution of WapA remains unknown, but it is important to understand the mechanism underlying the function of WapA. This study applied the fluorescence protein mCherry as a reporter gene to characterize the dynamic distribution of WapA in S. mutans via time-lapse and super-resolution fluorescence imaging. The results revealed interesting subcellular distribution patterns of WapA in single, dividing and long chains of S. mutans cells. It appears at the middle of the cell and moves to the poles as the cell grows and divides. In a cell chain, after each round of cell division, such dynamic relocation results in WapA distribution at the previous cell division sites, resulting in a pattern where WapA is located at the boundary of two adjacent cell pairs. This WapA distribution pattern corresponds to the breaking segmentation of wapA deletion cell chains. The dynamic relocation of WapA through the cell cycle increases our understanding of the mechanism of WapA in maintaining cell chain integrity and biofilm formation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. 14-3-3 proteins as signaling integration points for cell cycle control and apoptosis

    OpenAIRE

    Gardino, Alexandra K.; Yaffe, Michael B.

    2011-01-01

    14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent path...

  16. Host Proteins Determine MRSA Biofilm Structure and Integrity

    DEFF Research Database (Denmark)

    Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

    Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

  17. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  18. Integration of protein phosphorylation, acetylation, and methylation data sets to outline lung cancer signaling networks.

    Science.gov (United States)

    Grimes, Mark; Hall, Benjamin; Foltz, Lauren; Levy, Tyler; Rikova, Klarisa; Gaiser, Jeremiah; Cook, William; Smirnova, Ekaterina; Wheeler, Travis; Clark, Neil R; Lachmann, Alexander; Zhang, Bin; Hornbeck, Peter; Ma'ayan, Avi; Comb, Michael

    2018-05-22

    Protein posttranslational modifications (PTMs) have typically been studied independently, yet many proteins are modified by more than one PTM type, and cell signaling pathways somehow integrate this information. We coupled immunoprecipitation using PTM-specific antibodies with tandem mass tag (TMT) mass spectrometry to simultaneously examine phosphorylation, methylation, and acetylation in 45 lung cancer cell lines compared to normal lung tissue and to cell lines treated with anticancer drugs. This simultaneous, large-scale, integrative analysis of these PTMs using a cluster-filtered network (CFN) approach revealed that cell signaling pathways were outlined by clustering patterns in PTMs. We used the t-distributed stochastic neighbor embedding (t-SNE) method to identify PTM clusters and then integrated each with known protein-protein interactions (PPIs) to elucidate functional cell signaling pathways. The CFN identified known and previously unknown cell signaling pathways in lung cancer cells that were not present in normal lung epithelial tissue. In various proteins modified by more than one type of PTM, the incidence of those PTMs exhibited inverse relationships, suggesting that molecular exclusive "OR" gates determine a large number of signal transduction events. We also showed that the acetyltransferase EP300 appears to be a hub in the network of pathways involving different PTMs. In addition, the data shed light on the mechanism of action of geldanamycin, an HSP90 inhibitor. Together, the findings reveal that cell signaling pathways mediated by acetylation, methylation, and phosphorylation regulate the cytoskeleton, membrane traffic, and RNA binding protein-mediated control of gene expression. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  19. Actin Immobilization on Chitin for Purifying Myosin II: A Laboratory Exercise That Integrates Concepts of Molecular Cell Biology and Protein Chemistry

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    de Souza, Marcelle Gomes; Grossi, Andre Luiz; Pereira, Elisangela Lima Bastos; da Cruz, Carolina Oliveira; Mendes, Fernanda Machado; Cameron, Luiz Claudio; Paiva, Carmen Lucia Antao

    2008-01-01

    This article presents our experience on teaching biochemical sciences through an innovative approach that integrates concepts of molecular cell biology and protein chemistry. This original laboratory exercise is based on the preparation of an affinity chromatography column containing F-actin molecules immobilized on chitin particles for purifying…

  20. A Critical Role of TET1/2 Proteins in Cell-Cycle Progression of Trophoblast Stem Cells

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    Stephanie Chrysanthou

    2018-04-01

    Full Text Available Summary: The ten-eleven translocation (TET proteins are well known for their role in maintaining naive pluripotency of embryonic stem cells. Here, we demonstrate that, jointly, TET1 and TET2 also safeguard the self-renewal potential of trophoblast stem cells (TSCs and have partially redundant roles in maintaining the epithelial integrity of TSCs. For the more abundantly expressed TET1, we show that this is achieved by binding to critical epithelial genes, notably E-cadherin, which becomes hyper-methylated and downregulated in the absence of TET1. The epithelial-to-mesenchymal transition phenotype of mutant TSCs is accompanied by centrosome duplication and separation defects. Moreover, we identify a role of TET1 in maintaining cyclin B1 stability, thereby acting as facilitator of mitotic cell-cycle progression. As a result, Tet1/2 mutant TSCs are prone to undergo endoreduplicative cell cycles leading to the formation of polyploid trophoblast giant cells. Taken together, our data reveal essential functions of TET proteins in the trophoblast lineage. : TET proteins are well known for their role in pluripotency. Here, Hemberger and colleagues show that TET1 and TET2 are also critical for maintaining the epithelial integrity of trophoblast stem cells. TET1/2 ensure mitotic cell-cycle progression by stabilizing cyclin B1 and by regulating centrosome organization. These insights reveal the importance of TET proteins beyond their role in epigenome remodeling. Keywords: TET proteins, trophoblast stem cells, cell cycle, endoreduplication, self-renewal, mitosis, trophoblast giant cells, differentiation

  1. 14-3-3 Proteins in Guard Cell Signaling.

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    Cotelle, Valérie; Leonhardt, Nathalie

    2015-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

  2. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

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    Emily F A van 't Wout

    2015-06-01

    Full Text Available Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA. Efficient functioning of the endoplasmic reticulum (ER is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR. Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

  3. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

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    van ‘t Wout, Emily F. A.; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E.; Clarke, Hanna J.; Tommassen, Jan; Marciniak, Stefan J.; Hiemstra, Pieter S.

    2015-01-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  4. PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

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    Ines Teichert

    2014-09-01

    Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

  5. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

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    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  6. Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

    Directory of Open Access Journals (Sweden)

    Mickaël Desvaux

    2018-02-01

    Full Text Available The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria is formed of a cytoplasmic membrane (CM and a cell wall (CW. While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226, i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658, i.e., the lipoproteins. At the CW (GO: 0009275, cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed.

  7. Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression

    Directory of Open Access Journals (Sweden)

    Vandepoele Klaas

    2009-06-01

    Full Text Available Abstract Background Large-scale identification of the interrelationships between different components of the cell, such as the interactions between proteins, has recently gained great interest. However, unraveling large-scale protein-protein interaction maps is laborious and expensive. Moreover, assessing the reliability of the interactions can be cumbersome. Results In this study, we have developed a computational method that exploits the existing knowledge on protein-protein interactions in diverse species through orthologous relations on the one hand, and functional association data on the other hand to predict and filter protein-protein interactions in Arabidopsis thaliana. A highly reliable set of protein-protein interactions is predicted through this integrative approach making use of existing protein-protein interaction data from yeast, human, C. elegans and D. melanogaster. Localization, biological process, and co-expression data are used as powerful indicators for protein-protein interactions. The functional repertoire of the identified interactome reveals interactions between proteins functioning in well-conserved as well as plant-specific biological processes. We observe that although common mechanisms (e.g. actin polymerization and components (e.g. ARPs, actin-related proteins exist between different lineages, they are active in specific processes such as growth, cancer metastasis and trichome development in yeast, human and Arabidopsis, respectively. Conclusion We conclude that the integration of orthology with functional association data is adequate to predict protein-protein interactions. Through this approach, a high number of novel protein-protein interactions with diverse biological roles is discovered. Overall, we have predicted a reliable set of protein-protein interactions suitable for further computational as well as experimental analyses.

  8. Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

    Science.gov (United States)

    Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

    2011-01-01

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  9. Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

    Science.gov (United States)

    Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

    2018-04-01

    Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

  10. Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation

    Science.gov (United States)

    Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

    2017-01-01

    Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

  11. Piwi and potency: PIWI proteins in animal stem cells and regeneration.

    Science.gov (United States)

    van Wolfswinkel, Josien C

    2014-10-01

    PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

  12. Parvovirus B19 integration into human CD36+ erythroid progenitor cells.

    Science.gov (United States)

    Janovitz, Tyler; Wong, Susan; Young, Neal S; Oliveira, Thiago; Falck-Pedersen, Erik

    2017-11-01

    The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Dynamic changes in protein functional linkage networks revealed by integration with gene expression data.

    Directory of Open Access Journals (Sweden)

    Shubhada R Hegde

    2008-11-01

    Full Text Available Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data.

  14. Irradiation And Papillomavirus E2 Proteins On Hela Cells

    International Nuclear Information System (INIS)

    Abderrafi, B.

    2005-01-01

    Exposure to relatively high doses ionizing radiation activates cellular responses that impair cell survival. These responses, for which the p53 protein plays a central role, form the basis for cancer radiotherapy. However, the efficacy of radiation treatments on cell killing is often reduced as a consequence of the frequent inactivation of the p53 protein in cancer cells. Loss of p53 protein is associated with later stages of most human tumors and resistance to anticancer agents. Carcinomas are frequent malignant tumors in humans. The majority of cervical carcinomas are etiologically linked to the presence of HPV virus (Human Papillomavirus). In carcinoma tumor cells, as well as in their derived-cell lines such as HeLa cells, the p53 protein is generally not detected due to its degradation by the product of the HPV-associated oncogenic E6 gene. Another characteristic of HPV-positive cervical cancer cells is the loss of the regulatory viral E2 gene expression as a consequence of viral DNA integration into the cellular genome. Reintroduction of E2 expression in HeLa cells reactivates p53, due to a negative effect on the expression of E6 protein, with a concomitant arrest of cell proliferation at the phase G1 of the cell cycle and delay in cell division via the repression of E2F-target genes. To elucidate whether reactivation of p53 would improve the cell killing effect of ionizing radiation in cancer cells, we studied the combined effects of radiation and E2 expression on the cell cycle distribution in HeLa cells

  15. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

    Science.gov (United States)

    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  16. SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

    Science.gov (United States)

    Delattre, Marie; Balestra, Fernando R.; Blanchoud, Simon; Finger, Susanne; Knott, Graham; Müller-Reichert, Thomas; Gönczy, Pierre

    2014-01-01

    Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome. PMID:25412110

  17. SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

    Directory of Open Access Journals (Sweden)

    Lukas von Tobel

    2014-11-01

    Full Text Available Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD syndrome.

  18. Extracellular cell stress (heat shock) proteins-immune responses and disease: an overview.

    Science.gov (United States)

    Pockley, A Graham; Henderson, Brian

    2018-01-19

    Extracellular cell stress proteins are highly conserved phylogenetically and have been shown to act as powerful signalling agonists and receptors for selected ligands in several different settings. They also act as immunostimulatory 'danger signals' for the innate and adaptive immune systems. Other studies have shown that cell stress proteins and the induction of immune reactivity to self-cell stress proteins can attenuate disease processes. Some proteins (e.g. Hsp60, Hsp70, gp96) exhibit both inflammatory and anti-inflammatory properties, depending on the context in which they encounter responding immune cells. The burgeoning literature reporting the presence of stress proteins in a range of biological fluids in healthy individuals/non-diseased settings, the association of extracellular stress protein levels with a plethora of clinical and pathological conditions and the selective expression of a membrane form of Hsp70 on cancer cells now supports the concept that extracellular cell stress proteins are involved in maintaining/regulating organismal homeostasis and in disease processes and phenotype. Cell stress proteins, therefore, form a biologically complex extracellular cell stress protein network having diverse biological, homeostatic and immunomodulatory properties, the understanding of which offers exciting opportunities for delivering novel approaches to predict, identify, diagnose, manage and treat disease.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'. © 2017 The Author(s).

  19. Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

    International Nuclear Information System (INIS)

    Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

    1987-01-01

    The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

  20. The roles of USH1 proteins and PDZ domain-containing USH proteins in USH2 complex integrity in cochlear hair cells.

    Science.gov (United States)

    Zou, Junhuang; Chen, Qian; Almishaal, Ali; Mathur, Pranav Dinesh; Zheng, Tihua; Tian, Cong; Zheng, Qing Y; Yang, Jun

    2017-02-01

    Usher syndrome (USH) is the most common cause of inherited deaf-blindness, manifested as USH1, USH2 and USH3 clinical types. The protein products of USH2 causative and modifier genes, USH2A, ADGRV1, WHRN and PDZD7, interact to assemble a multiprotein complex at the ankle link region of the mechanosensitive stereociliary bundle in hair cells. Defects in this complex cause stereociliary bundle disorganization and hearing loss. The four USH2 proteins also interact in vitro with USH1 proteins including myosin VIIa, USH1G (SANS), CIB2 and harmonin. However, it is unclear whether the interactions between USH1 and USH2 proteins occur in vivo and whether USH1 proteins play a role in USH2 complex assembly in hair cells. In this study, we identified a novel interaction between myosin VIIa and PDZD7 by FLAG pull-down assay. We further investigated the role of the above-mentioned four USH1 proteins in the cochlear USH2 complex assembly using USH1 mutant mice. We showed that only myosin VIIa is indispensable for USH2 complex assembly at ankle links, indicating the potential transport and/or anchoring role of myosin VIIa for USH2 proteins in hair cells. However, myosin VIIa is not required for USH2 complex assembly in photoreceptors. We further showed that, while PDZ protein harmonin is not involved, its paralogous USH2 proteins, PDZD7 and whirlin, function synergistically in USH2 complex assembly in cochlear hair cells. In summary, our studies provide novel insight into the functional relationship between USH1 and USH2 proteins in the cochlea and the retina as well as the disease mechanisms underlying USH1 and USH2. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Tumor cell surface proteins

    International Nuclear Information System (INIS)

    Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

    1982-01-01

    Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

  2. A multi-landing pad DNA integration platform for mammalian cell engineering

    Science.gov (United States)

    Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

    2018-01-01

    Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

  3. Production of animal and vegetable proteins: an integrated thermal approach

    Energy Technology Data Exchange (ETDEWEB)

    Kesari, J P; Bonvehi, F; De Saint-Salvy, A; Miquel, J F

    1984-01-01

    For the optimization of our integrated farm, theoretical models using a microcomputer and experimental tests to verify these models were carried out on two research units. A test cell integrated with a greenhouse and a rock bed and a standard rock bed coupled with solar air collectors. A complete wooden house has been constructed and experimented in a remote village 200 km north of Toulouse as part of a demonstration unit. The geese and the Lemna minor (duckweed) have been selected as an animal and as a vegetable for the protein production. Some of the experimental results are reported.

  4. Nucleophosmin is required for DNA integrity and p19Arf protein stability

    DEFF Research Database (Denmark)

    Colombo, Emanuela; Bonetti, Paola; Lazzerini Denchi, Eros

    2005-01-01

    , such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM....

  5. The Unfolded Protein Response and Cell Fate Control.

    Science.gov (United States)

    Hetz, Claudio; Papa, Feroz R

    2018-01-18

    The secretory capacity of a cell is constantly challenged by physiological demands and pathological perturbations. To adjust and match the protein-folding capacity of the endoplasmic reticulum (ER) to changing secretory needs, cells employ a dynamic intracellular signaling pathway known as the unfolded protein response (UPR). Homeostatic activation of the UPR enforces adaptive programs that modulate and augment key aspects of the entire secretory pathway, whereas maladaptive UPR outputs trigger apoptosis. Here, we discuss recent advances into how the UPR integrates information about the intensity and duration of ER stress stimuli in order to control cell fate. These findings are timely and significant because they inform an evolving mechanistic understanding of a wide variety of human diseases, including diabetes mellitus, neurodegeneration, and cancer, thus opening up the potential for new therapeutic modalities to treat these diverse diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Plant virus cell-to-cell movement is not dependent on the transmembrane disposition of its movement protein.

    Science.gov (United States)

    Martínez-Gil, Luis; Sánchez-Navarro, Jesús A; Cruz, Antonio; Pallás, Vicente; Pérez-Gil, Jesús; Mingarro, Ismael

    2009-06-01

    The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.

  7. A scored human protein-protein interaction network to catalyze genomic interpretation

    DEFF Research Database (Denmark)

    Li, Taibo; Wernersson, Rasmus; Hansen, Rasmus B

    2017-01-01

    Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap,......Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (In...

  8. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

  9. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Imaging protein-protein interactions in living cells

    NARCIS (Netherlands)

    Hink, M.A.; Bisseling, T.; Visser, A.J.W.G.

    2002-01-01

    The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide

  11. Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation

    International Nuclear Information System (INIS)

    Mannioui, Abdelkrim; Schiffer, Cecile; Felix, Nathalie

    2004-01-01

    We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression

  12. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C......-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat......The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared...

  13. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

    Directory of Open Access Journals (Sweden)

    Ming Wang

    Full Text Available Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

  14. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

  15. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

    Science.gov (United States)

    Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  16. SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

    Science.gov (United States)

    Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G; Fu, Xiang-Dong

    2009-07-10

    SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

  17. A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

    Science.gov (United States)

    Niphakis, Micah J; Lum, Kenneth M; Cognetta, Armand B; Correia, Bruno E; Ichu, Taka-Aki; Olucha, Jose; Brown, Steven J; Kundu, Soumajit; Piscitelli, Fabiana; Rosen, Hugh; Cravatt, Benjamin F

    2015-06-18

    Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

    Science.gov (United States)

    Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

    2018-03-01

    During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

    Energy Technology Data Exchange (ETDEWEB)

    Mushegian, Arcady R., E-mail: mushegian2@gmail.com [Division of Molecular and Cellular Biosciences, National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230 (United States); Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es [Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, 46022 València (Spain); The Santa Fe Institute, Santa Fe, NM 87501 (United States)

    2015-02-15

    Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.

  20. InSilico Proteomics System: Integration and Application of Protein and Protein-Protein Interaction Data using Microsoft .NET

    Directory of Open Access Journals (Sweden)

    Straßer Wolfgang

    2006-12-01

    Full Text Available In the last decades, biological databases became the major knowledge resource for researchers in the field of molecular biology. The distribution of information among these databases is one of the major problems. An overview about the subject area of data access and representation of protein and protein-protein interaction data within public biological databases is described. For a comprehensive and consistent way of searching and analysing integrated protein and protein-protein interaction data, the InSilico Proteomics (ISP project has been initiated. Its three main objectives are (1 to provide an integrated knowledge pool for data investigation and global network analysis functions for a better understanding of a cell’s interactome, (2 employment of public data for plausibility analysis and validation of in-house experimental data and (3 testing the applicability of Microsoft’s .NET architecture for bioinformatics applications. Data integrated into the ISP database can be queried through the Web portal PRIMOS (PRotein Interaction and MOlecule Search which is freely available at http://biomis.fh-hagenberg.at/isp/primos.

  1. Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

    Science.gov (United States)

    Yang, Jae-Seong; Kwon, Oh Sung; Kim, Sanguk; Jang, Sung Key

    2013-01-01

    Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets. PMID:23593195

  2. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Kim Yeon-Gu

    2012-05-01

    Full Text Available Abstract Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES was introduced for using two green fluorescence protein (GFP fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

  3. Integration of statistical modeling and high-content microscopy to systematically investigate cell-substrate interactions.

    Science.gov (United States)

    Chen, Wen Li Kelly; Likhitpanichkul, Morakot; Ho, Anthony; Simmons, Craig A

    2010-03-01

    Cell-substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3-144 kPa) and adhesion protein concentration (7-520 microg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell-material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell-material interfaces. Copyright 2009 Elsevier Ltd. All rights reserved.

  4. The GPI-anchored protein Ecm33 is vital for conidiation, cell wall integrity, and multi-stress tolerance of two filamentous entomopathogens but not for virulence.

    Science.gov (United States)

    Chen, Ying; Zhu, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2014-06-01

    Ecm33 is one of several glycosylphosphatidylinositol (GPI)-anchored proteins. This protein is known to be involved in fungal cell wall integrity, but its contribution to multi-stress tolerance is largely unknown. Here we characterized the functions of two Ecm33 orthologues, i.e., Bbecm33 in Beauveria bassiana and Mrecm33 in Metarhizium robertsii. Bbecm33 and Mrecm33 were both confirmed as GPI-anchored cell wall proteins in immunogold localization. Single-gene disruptions of Bbecm33 and Mrecm33 caused slight growth defects, but conidial yield decreased much more in ΔBbecm33 (76 %) than in ΔMrecm33 (42 %), accompanied with significant reductions of intracellular mannitol and trehalose contents in both mutants and weakened cell walls in ΔBbecm33 only. Consequently, ΔBbecm33 was far more sensitive to the cell wall-perturbating agents Congo red and sodium dodecyl sulfate (SDS) than ΔMrecm33, which showed null response to SDS. Both deletion mutants became significantly more sensitive to two oxidants (menadione and H2O2), two fungicides (carbendazim and ethirimol), osmotic salt NaCl, and Ca(2+) during growth despite some degrees of differences in their sensitivities to the chemical stressors. Strikingly, conidial UV-B resistance decreased by 55 % in ΔBbecm33 but was unaffected in ΔMrecm33, unlike a similar decrease (25-28 %) of conidial thermotolerance in both. All the changes were restored to wild-type levels by gene complementation through ectopic gene integration in each fungus. However, neither ΔBbecm33 nor ΔMrecm33 showed a significant change in virulence to a susceptible insect host. Our results indicate that Bbecm33 and Mrecm33 contribute differentially to the conidiation and multi-stress tolerance of B. bassiana and M. robertsii.

  5. Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

    Science.gov (United States)

    Sivagurunathan, Suganya; Palanisamy, Karthikka; Arunachalam, Jayamuruga Pandian; Chidambaram, Subbulakshmi

    2017-03-01

    PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

  6. Communication Between the Cell Membrane and the Nucleus: Role of Protein Compartmentalization

    Energy Technology Data Exchange (ETDEWEB)

    Lelievre, Sophie A; Bissell, Mina J

    1998-10-21

    Understanding how the information is conveyed from outside to inside the cell is a critical challenge for all biologists involved in signal transduction. The flow of information initiated by cell-cell and cell-extracellular matrix contacts is mediated by the formation of adhesion complexes involving multiple proteins. Inside adhesion complexes, connective membrane skeleton (CMS) proteins are signal transducers that bind to adhesion molecules, organize the cytoskeleton, and initiate biochemical cascades. Adhesion complex-mediated signal transduction ultimately directs the formation of supramolecular structures in the cell nucleus, as illustrated by the establishment of multi complexes of DNA-bound transcription factors, and the redistribution of nuclear structural proteins to form nuclear subdomains. Recently, several CMS proteins have been observed to travel to the cell nucleus, suggesting a distinctive role for these proteins in signal transduction. This review focuses on the nuclear translocation of structural signal transducers of the membrane skeleton and also extends our analysis to possible translocation of resident nuclear proteins to the membrane skeleton. This leads us to envision the communication between spatially distant cellular compartments (i.e., membrane skeleton and cell nucleus) as a bidirectional flow of information (a dynamic reciprocity) based on subtle multilevel structural and biochemical equilibria. At one level, it is mediated by the interaction between structural signal transducers and their binding partners, at another level it may be mediated by the balance and integration of signal transducers in different cellular compartments.

  7. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  8. Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

    Directory of Open Access Journals (Sweden)

    Jeong Y

    2015-11-01

    Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

  9. CellMap visualizes protein-protein interactions and subcellular localization

    Science.gov (United States)

    Dallago, Christian; Goldberg, Tatyana; Andrade-Navarro, Miguel Angel; Alanis-Lobato, Gregorio; Rost, Burkhard

    2018-01-01

    Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers. PMID:29497493

  10. VP22 herpes simplex virus protein can transduce proteins into stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gabanyi, I.; Lojudice, F.H.; Kossugue, P.M. [Centro de Terapia Celular e Molecular, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP (Brazil); Rebelato, E. [Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Demasi, M.A.; Sogayar, M.C. [Centro de Terapia Celular e Molecular, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP (Brazil)

    2013-02-01

    The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.

  11. VP22 herpes simplex virus protein can transduce proteins into stem cells

    International Nuclear Information System (INIS)

    Gabanyi, I.; Lojudice, F.H.; Kossugue, P.M.; Rebelato, E.; Demasi, M.A.; Sogayar, M.C.

    2013-01-01

    The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations

  12. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Say Kong Ng

    2013-04-01

    Full Text Available From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX amplification technology or the glutamine synthetase (GS system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

  13. New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Malcová, Ivana; Farkasovky, M.; Senohrábková, Lenka; Vašicová, Pavla; Hašek, Jiří

    2016-01-01

    Roč. 16, č. 3 (2016), fow027 ISSN 1567-1356 R&D Projects: GA ČR GAP305/12/0480; GA ČR(CZ) GA16-05497S Institutional support: RVO:61388971 Keywords : fluorescent proteins * dominant selectable markers * Integrative cassettes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.299, year: 2016

  14. Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip

    DEFF Research Database (Denmark)

    Matos, T.; Senkbeil, Silja; Mendonça, A.

    2013-01-01

    technique has been developed which is based on exposing E. coli cells to low voltages to allow extraction of nucleic acids and proteins. The flow-through electropermeability chip used consists of a microfluidic channel with integrated gold electrodes that promote cell envelope channel formation at low...

  15. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

    DEFF Research Database (Denmark)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim

    2017-01-01

    , as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion...... of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various......-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB....

  16. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

    Science.gov (United States)

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

    2017-07-01

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

  17. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    Science.gov (United States)

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  18. Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development.

    Science.gov (United States)

    Becker, Emmanuelle; Liu, Yuchen; Lardenois, Aurélie; Walther, Thomas; Horecka, Joe; Stuparevic, Igor; Law, Michael J; Lavigne, Régis; Evrard, Bertrand; Demougin, Philippe; Riffle, Michael; Strich, Randy; Davis, Ronald W; Pineau, Charles; Primig, Michael

    2015-04-24

    Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/α strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis. Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field-which investigate haploid yeast strains-because only diploid cells can undergo meiotic development

  19. Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

    Science.gov (United States)

    Vogl, Thomas; Gebbie, Leigh; Palfreyman, Robin W; Speight, Robert

    2018-03-15

    Saccharomyces cerevisiae , expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii ). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains. Copyright © 2018 American Society for Microbiology.

  20. Electrochemical protein cleavage in a microfluidic cell with integrated boron doped diamond electrodes

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Zhang, Tao; Ma, Liwei; Odijk, Mathieu; Olthuis, Wouter; Permentier, Hjalmar P.; Bischoff, Rainer P.H.; van den Berg, Albert

    2015-01-01

    We present a microfluidic electrochemical cell with integrated boron doped diamond (BDD) electrodes which is designed for high electrochemical conversion efficiencies. With our newest developments, we aim to exploit the benefits of BDD as a novel electrode material to conduct tyrosine- and

  1. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  2. The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

    Science.gov (United States)

    Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

    2017-08-01

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.

    Science.gov (United States)

    Kateja, Nikhil; Agarwal, Harshit; Hebbi, Vishwanath; Rathore, Anurag S

    2017-07-01

    Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017. © 2016 American Institute of Chemical Engineers.

  4. Cell signaling heterogeneity is modulated by both cell-intrinsic and -extrinsic mechanisms: An integrated approach to understanding targeted therapy.

    Science.gov (United States)

    Kim, Eunjung; Kim, Jae-Young; Smith, Matthew A; Haura, Eric B; Anderson, Alexander R A

    2018-03-01

    During the last decade, our understanding of cancer cell signaling networks has significantly improved, leading to the development of various targeted therapies that have elicited profound but, unfortunately, short-lived responses. This is, in part, due to the fact that these targeted therapies ignore context and average out heterogeneity. Here, we present a mathematical framework that addresses the impact of signaling heterogeneity on targeted therapy outcomes. We employ a simplified oncogenic rat sarcoma (RAS)-driven mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway in lung cancer as an experimental model system and develop a network model of the pathway. We measure how inhibition of the pathway modulates protein phosphorylation as well as cell viability under different microenvironmental conditions. Training the model on this data using Monte Carlo simulation results in a suite of in silico cells whose relative protein activities and cell viability match experimental observation. The calibrated model predicts distributional responses to kinase inhibitors and suggests drug resistance mechanisms that can be exploited in drug combination strategies. The suggested combination strategies are validated using in vitro experimental data. The validated in silico cells are further interrogated through an unsupervised clustering analysis and then integrated into a mathematical model of tumor growth in a homogeneous and resource-limited microenvironment. We assess posttreatment heterogeneity and predict vast differences across treatments with similar efficacy, further emphasizing that heterogeneity should modulate treatment strategies. The signaling model is also integrated into a hybrid cellular automata (HCA) model of tumor growth in a spatially heterogeneous microenvironment. As a proof of concept, we simulate tumor responses to targeted therapies in a spatially segregated tissue structure containing tumor

  5. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

    Directory of Open Access Journals (Sweden)

    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  6. Integration by cell algorithm for Slater integrals in a spline basis

    International Nuclear Information System (INIS)

    Qiu, Y.; Fischer, C.F.

    1999-01-01

    An algorithm for evaluating Slater integrals in a B-spline basis is introduced. Based on the piecewise property of the B-splines, the algorithm divides the two-dimensional (r 1 , r 2 ) region into a number of rectangular cells according to the chosen grid and implements the two-dimensional integration over each individual cell using Gaussian quadrature. Over the off-diagonal cells, the integrands are separable so that each two-dimensional cell-integral is reduced to a product of two one-dimensional integrals. Furthermore, the scaling invariance of the B-splines in the logarithmic region of the chosen grid is fully exploited such that only some of the cell integrations need to be implemented. The values of given Slater integrals are obtained by assembling the cell integrals. This algorithm significantly improves the efficiency and accuracy of the traditional method that relies on the solution of differential equations and renders the B-spline method more effective when applied to multi-electron atomic systems

  7. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid

    OpenAIRE

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-01-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane α-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lip...

  8. Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

    Directory of Open Access Journals (Sweden)

    Carmen I. Nussbaum-Krammer

    2014-01-01

    Full Text Available Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.

  9. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Directory of Open Access Journals (Sweden)

    Alexander Zhyvoloup

    2017-07-01

    Full Text Available HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  10. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

    Science.gov (United States)

    Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

    2017-07-01

    HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

  11. Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

    Science.gov (United States)

    Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

    2015-01-01

    Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria

    Directory of Open Access Journals (Sweden)

    Sachith D. Gunasinghe

    2017-05-01

    Full Text Available Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.

  13. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

    Science.gov (United States)

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

    2017-06-01

    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  14. Mental retardation-related protease, motopsin (prss12), binds to the BRICHOS domain of the integral membrane protein 2a.

    Science.gov (United States)

    Mitsui, Shinichi; Osako, Yoji; Yuri, Kazunari

    2014-01-01

    Motopsin (prss12), a mosaic serine protease secreted by neuronal cells, is believed to be important for cognitive function, as the loss of its function causes severe nonsyndromic mental retardation. To understand the molecular role of motopsin, we identified the integral membrane protein 2a (Itm2a) as a motopsin-interacting protein using a yeast two-hybrid system. A pull-down assay showed that the BRICHOS domain of Itm2a was essential for this interaction. Motopsin and Itm2a co-localized in COS cells and in cultured neurons when transiently expressed in these cells. Both proteins were co-immunoprecipitated from lysates of these transfected COS cells. Itm2a was strongly detected in a brain lysate prepared between postnatal day 0 and 10, during which period motopsin protein was also enriched in the brain. Immunohistochemistry detected Itm2a as patchy spots along endothelial cells of brain capillaries (which also expressed myosin II regulatory light chain [RLC]), and on glial fibrillary acidic protein (GFAP)-positive processes in the developing cerebral cortex. The data raise the possibility that secreted motopsin interacts with endothelial cells in the developing brain. © 2013 International Federation for Cell Biology.

  15. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  16. Cell Culture Systems To Study Human Herpesvirus 6A/B Chromosomal Integration.

    Science.gov (United States)

    Gravel, Annie; Dubuc, Isabelle; Wallaschek, Nina; Gilbert-Girard, Shella; Collin, Vanessa; Hall-Sedlak, Ruth; Jerome, Keith R; Mori, Yasuko; Carbonneau, Julie; Boivin, Guy; Kaufer, Benedikt B; Flamand, Louis

    2017-07-15

    Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39 , U90 , and U100 , without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state. IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the

  17. The cell wall protein Ecm33 of Candida albicans is involved in chronological life span, morphogenesis, cell wall regeneration, stress tolerance and host-cell interaction

    Directory of Open Access Journals (Sweden)

    Ana eGil-Bona

    2016-02-01

    Full Text Available Ecm33 is a glycosylphosphatidylinositol (GPI-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the cell wall integrity pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a veil growth, never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

  18. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  19. Protein diffusion in plant cell plasma membranes: the cell-wall corral.

    Science.gov (United States)

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  20. Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

    Science.gov (United States)

    Luo, Wentian; Galvan, Daniel L; Woodard, Lauren E; Dorset, Dan; Levy, Shawn; Wilson, Matthew H

    2017-08-21

    Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  1. Diffusion of Integral Membrane Proteins in Protein-Rich Membranes

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Metzler, Ralf

    2017-01-01

    of being protein-poor, native cell membranes are extremely crowded with proteins. On the basis of extensive molecular simulations, we here demonstrate that protein crowding of the membrane at physiological levels leads to deviations from the SD relation and to the emergence of a stronger Stokes......-like dependence D ∝ 1/R. We propose that this 1/R law mainly arises due to geometrical factors: smaller proteins are able to avoid confinement effects much better than their larger counterparts. The results highlight that the lateral dynamics in the crowded setting found in native membranes is radically different......The lateral diffusion of embedded proteins along lipid membranes in protein-poor conditions has been successfully described in terms of the Saffman-Delbrück (SD) model, which predicts that the protein diffusion coefficient D is weakly dependent on its radius R as D ∝ ln(1/R). However, instead...

  2. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Science.gov (United States)

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Protein diffusion in plant cell plasma membranes: The cell-wall corral

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

    Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  4. Cell-free protein synthesis in micro compartments: building a minimal cell from biobricks.

    Science.gov (United States)

    Jia, Haiyang; Heymann, Michael; Bernhard, Frank; Schwille, Petra; Kai, Lei

    2017-10-25

    The construction of a minimal cell that exhibits the essential characteristics of life is a great challenge in the field of synthetic biology. Assembling a minimal cell requires multidisciplinary expertise from physics, chemistry and biology. Scientists from different backgrounds tend to define the essence of 'life' differently and have thus proposed different artificial cell models possessing one or several essential features of living cells. Using the tools and methods of molecular biology, the bottom-up engineering of a minimal cell appears in reach. However, several challenges still remain. In particular, the integration of individual sub-systems that is required to achieve a self-reproducing cell model presents a complex optimization challenge. For example, multiple self-organisation and self-assembly processes have to be carefully tuned. We review advances and developments of new methods and techniques, for cell-free protein synthesis as well as micro-fabrication, for their potential to resolve challenges and to accelerate the development of minimal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  6. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    International Nuclear Information System (INIS)

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J.

    2006-01-01

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1α associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome

  7. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    International Nuclear Information System (INIS)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun; Lim, Chaeseung; Kim, Jungho; Cha, Dae Ryong; Oh, Junseo

    2012-01-01

    Highlights: ► We designed novel recombinant albumin-RBP fusion proteins. ► Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). ► Fusion proteins are successfully internalized into and inactivate PSCs. ► RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I–III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin domain III (R-III) and albumin domain I -RBP-albumin III (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises of stellate cell inactivation-inducing moiety and targeting moiety, which may lead to the development of effective anti

  8. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

  9. Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

    Science.gov (United States)

    Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

    2010-05-01

    High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

  10. Detection of DNA strand breaks in mammalian cells using the radioresistant bacterium PprA protein

    International Nuclear Information System (INIS)

    Satoh, Katsuya; Wada, Seiichi; Narumi, Issay; Kikuchi, Masahiro; Funayama, Tomoo; Kobayashi, Yasuhiko

    2003-01-01

    We have previously found that the PprA protein from Deinococcus radiodurans possesses ability to recognize DNA carrying strand breaks. In the present study, we attempted to visualize radiation-induced DNA strand breaks with PprA protein using immunofluorescence technique to elucidate the DNA damage response mechanism in mammalian cultured cells. As a result, colocalization of Cy2 and DAPI fluorescent signals was observed. This observation suggests that DNA strand breaks in the nucleus of CHO-K1 cells were effectively detected using the PprA protein. The amount of DNA strand breaks (integrated density of Cy2 fluorescent signals) was increased with the increase in the radiation dose. (author)

  11. Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells

    International Nuclear Information System (INIS)

    Silverman, J.A.; Mehta, J.; Brocher, S.; Amenta, J.S.

    1985-01-01

    Previous studies on protein turnover in 3 H-labelled L-cell cultures have shown recovery of total 3 H at the end of a three-day experiment to be always significantly in excess of the 3 H recovered at the beginning of the experiment. A number of possible sources for this error in measuring radioactivity in cell proteins has been reviewed. 3 H-labelled proteins, when dissolved in NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. Furthermore, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed. (author)

  12. Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

    2012-03-01

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. The master two-dimensional gel database of human AMA cell proteins: towards linking protein and genome sequence and mapping information (update 1991)

    DEFF Research Database (Denmark)

    Celis, J E; Leffers, H; Rasmussen, H H

    1991-01-01

    autoantigens" and "cDNAs". For convenience we have included an alphabetical list of all known proteins recorded in this database. In the long run, the main goal of this database is to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture......The master two-dimensional gel database of human AMA cells currently lists 3801 cellular and secreted proteins, of which 371 cellular polypeptides (306 IEF; 65 NEPHGE) were added to the master images during the last 10 months. These include: (i) very basic and acidic proteins that do not focus...

  14. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    Science.gov (United States)

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  15. Passive acquisition of leukocyte proteins is associated with changes in phosphorylation of cellular proteins and cell-cell adhesion properties.

    OpenAIRE

    Tabibzadeh, S. S.; Kong, Q. F.; Kapur, S.

    1994-01-01

    In this report, we show that interaction of neoplastic epithelial cells with vesicles derived from leukocytes results in passive acquisition by tumor cells of a diverse group of leukocyte proteins. Vesicles shed from leukocytes were heterogeneous and exhibited the specific proteins expressed on leukocyte subsets. Accordingly, epithelial cells differentially acquired leukocyte proteins associated with vesicles. Ultrastructural localization demonstrated that acquired proteins were associated wi...

  16. Knockout of MIMP protein in lactobacillus plantarum lost its regulation of intestinal permeability on NCM460 epithelial cells through the zonulin pathway.

    Science.gov (United States)

    Liu, Zhihua; Kang, Liang; Li, Chao; Tong, Chao; Huang, Meijin; Zhang, Xingwei; Huang, Nanqi; Moyer, Mary Pat; Qin, Huanlong; Wang, Jianping

    2014-10-03

    Previous studies indicated that the micro integral membrane protein located within the media place of the integral membrane protein of Lactobacillus plantarum CGMCC 1258 had protective effects against the intestinal epithelial injury. In our study, we mean to establish micro integral membrane protein -knockout Lactobacillus plantarum (LPKM) to investigate the change of its protective effects and verify the role of micro integral membrane protein on protection of normal intestinal barrier function. Binding assay and intestinal permeability were performed to verify the protective effects of micro integral membrane protein on intestinal permeability in vitro and in vivo. Molecular mechanism was also determined as the zonulin pathway. Clinical data were also collected for further verification of relationship between zonulin level and postoperative septicemia. LPKM got decreased inhibition of EPEC adhesion to NCM460 cells. LPKM had lower ability to alleviate the decrease of intestinal permeability induced by enteropathogenic-e.coli, and prevent enteropathogenic-e.coli -induced increase of zonulin expression. Overexpression of zonulin lowered the intestinal permeability regulated by Lactobacillus plantarum. There was a positive correlation between zonulin level and postoperative septicemia. Therefore, micro integral membrane protein could be necessary for the protective effects of Lactobacillus plantarum on intestinal barrier. MIMP might be a positive factor for Lactobacillus plantarum to protect the intestinal epithelial cells from injury, which could be related to the zonulin pathway.

  17. A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division

    Science.gov (United States)

    Cruz-Ramírez, Alfredo; Díaz-Triviño, Sara; Blilou, Ikram; Grieneisen, Verônica A.; Sozzani, Rosangela; Zamioudis, Christos; Miskolczi, Pál; Nieuwland, Jeroen; Benjamins, René; Dhonukshe, Pankaj; Caballero-Pérez, Juan; Horvath, Beatrix; Long, Yuchen; Mähönen, Ari Pekka; Zhang, Hongtao; Xu, Jian; Murray, James A.H.; Benfey, Philip N.; Bako, Laszlo; Marée, Athanasius F.M.; Scheres, Ben

    2012-01-01

    SUMMARY In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a “flip flop” that constrains asymmetric cell division to the stem cell region. PMID:22921914

  18. Recombinant fusion protein of albumin-retinol binding protein inactivates stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Soyoung; Park, Sangeun; Kim, Suhyun [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of); Lim, Chaeseung [Department of Laboratory Medicine, Korea University Guro Hospital, Seoul 152-703 (Korea, Republic of); Kim, Jungho [Department of Life Science, Sogang University, Seoul 121-742 (Korea, Republic of); Cha, Dae Ryong [Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Gyeonggi do 425-020 (Korea, Republic of); Oh, Junseo, E-mail: ohjs@korea.ac.kr [Laboratory of Cellular Oncology, Korea University Graduate School of Medicine, Ansan, Gyeonggi do 425-707 (Korea, Republic of)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We designed novel recombinant albumin-RBP fusion proteins. Black-Right-Pointing-Pointer Expression of fusion proteins inactivates pancreatic stellate cells (PSCs). Black-Right-Pointing-Pointer Fusion proteins are successfully internalized into and inactivate PSCs. Black-Right-Pointing-Pointer RBP moiety mediates cell specific uptake of fusion protein. -- Abstract: Quiescent pancreatic- (PSCs) and hepatic- (HSCs) stellate cells store vitamin A (retinol) in lipid droplets via retinol binding protein (RBP) receptor and, when activated by profibrogenic stimuli, they transform into myofibroblast-like cells which play a key role in the fibrogenesis. Despite extensive investigations, there is, however, currently no appropriate therapy available for tissue fibrosis. We previously showed that the expression of albumin, composed of three homologous domains (I-III), inhibits stellate cell activation, which requires its high-affinity fatty acid-binding sites asymmetrically distributed in domain I and III. To attain stellate cell-specific uptake, albumin (domain I/III) was coupled to RBP; RBP-albumin{sup domain} {sup III} (R-III) and albumin{sup domain} {sup I}-RBP-albumin{sup III} (I-R-III). To assess the biological activity of fusion proteins, cultured PSCs were used. Like wild type albumin, expression of R-III or I-R-III in PSCs after passage 2 (activated PSCs) induced phenotypic reversal from activated to fat-storing cells. On the other hand, R-III and I-R-III, but not albumin, secreted from transfected 293 cells were successfully internalized into and inactivated PSCs. FPLC-purified R-III was found to be internalized into PSCs via caveolae-mediated endocytosis, and its efficient cellular uptake was also observed in HSCs and podocytes among several cell lines tested. Moreover, tissue distribution of intravenously injected R-III was closely similar to that of RBP. Therefore, our data suggest that albumin-RBP fusion protein comprises

  19. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

    NARCIS (Netherlands)

    Peifer, Martin; Fernandez-Cuesta, Lynnette; Sos, Martin L.; George, Julie; Seidel, Danila; Kasper, Lawryn H.; Plenker, Dennis; Leenders, Frauke; Sun, Ruping; Zander, Thomas; Menon, Roopika; Koker, Mirjam; Dahmen, Ilona; Mueller, Christian; Di Cerbo, Vincenzo; Schildhaus, Hans-Ulrich; Altmueller, Janine; Baessmann, Ingelore; Becker, Christian; de Wilde, Bram; Vandesompele, Jo; Boehm, Diana; Ansen, Sascha; Gabler, Franziska; Wilkening, Ines; Heynck, Stefanie; Heuckmann, Johannes M.; Lu, Xin; Carter, Scott L.; Cibulskis, Kristian; Banerji, Shantanu; Getz, Gad; Park, Kwon-Sik; Rauh, Daniel; Gruetter, Christian; Fischer, Matthias; Pasqualucci, Laura; Wright, Gavin; Wainer, Zoe; Russell, Prudence; Petersen, Iver; Chen, Yuan; Stoelben, Erich; Ludwig, Corinna; Schnabel, Philipp; Hoffmann, Hans; Muley, Thomas; Brockmann, Michael; Engel-Riedel, Walburga; Muscarella, Lucia A.; Fazio, Vito M.; Groen, Harry; Timens, Wim; Sietsma, Hannie; Thunnissen, Erik; Smit, Egbert; Heideman, Danielle A. M.; Snijders, Peter J. F.; Cappuzzo, Federico; Ligorio, Claudia; Damiani, Stefania; Field, John; Solberg, Steinar; Brustugun, Odd Terje; Lund-Iversen, Marius; Saenger, Joerg; Clement, Joachim H.; Soltermann, Alex; Moch, Holger; Weder, Walter; Solomon, Benjamin; Soria, Jean-Charles; Validire, Pierre; Besse, Benjamin; Brambilla, Elisabeth; Brambilla, Christian; Lantuejoul, Sylvie; Lorimier, Philippe; Schneider, Peter M.; Hallek, Michael; Pao, William; Meyerson, Matthew; Sage, Julien; Shendure, Jay; Schneider, Robert; Buettner, Reinhard; Wolf, Juergen; Nuernberg, Peter; Perner, Sven; Heukamp, Lukas C.; Brindle, Paul K.; Haas, Stefan; Thomas, Roman K.

    2012-01-01

    Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis(1-3). We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 +/- 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated

  20. Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

    Science.gov (United States)

    Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

    2015-04-03

    RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein

    International Nuclear Information System (INIS)

    Shipp, M.A.; Richardson, N.E.; Sayre, P.H.; Brown, N.R.; Masteller, E.L.; Clayton, L.K.; Ritz, J.; Reinherz, E.L.

    1988-01-01

    Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate the primary structure of CALLA, the authors purified the protein to homogeneity, obtained the NH 2 -terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line λgt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NM 2 -terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA + cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types

  2. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    Science.gov (United States)

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013.

  3. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  4. The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling.

    Science.gov (United States)

    Feng, Wei; Kita, Daniel; Peaucelle, Alexis; Cartwright, Heather N; Doan, Vinh; Duan, Qiaohong; Liu, Ming-Che; Maman, Jacob; Steinhorst, Leonie; Schmitz-Thom, Ina; Yvon, Robert; Kudla, Jörg; Wu, Hen-Ming; Cheung, Alice Y; Dinneny, José R

    2018-03-05

    Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. The Proteins API: accessing key integrated protein and genome information.

    Science.gov (United States)

    Nightingale, Andrew; Antunes, Ricardo; Alpi, Emanuele; Bursteinas, Borisas; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd; Martin, Maria

    2017-07-03

    The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to 'talk' to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at (http://www.ebi.ac.uk/proteins/api/doc). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. ProNormz--an integrated approach for human proteins and protein kinases normalization.

    Science.gov (United States)

    Subramani, Suresh; Raja, Kalpana; Natarajan, Jeyakumar

    2014-02-01

    The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein-protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. 17-Allylamino-17-demethoxygeldanamycin induces downregulation of critical Hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells

    International Nuclear Information System (INIS)

    Karkoulis, Panagiotis K; Stravopodis, Dimitrios J; Margaritis, Lukas H; Voutsinas, Gerassimos E

    2010-01-01

    17-Allylamino-17-demethoxygeldanamycin (17-AAG), a benzoquinone ansamycin antibiotic, specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone that maintains the structural and functional integrity of various protein clients involved in cellular signaling. In this study, we have investigated the effect of 17-AAG on the regulation of Hsp90-dependent signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cell lines. We have used MTT-based assays, FACS analysis, Western blotting, semi-quantitative RT-PCR, immunocytochemistry and scratch-wound assay in RT4, RT112 and T24 human urinary bladder cancer cell lines. We have demonstrated that, upon 17-AAG treatment, bladder cancer cells are arrested in the G1 phase of the cell cycle and eventually undergo apoptotic cell death in a dose-dependent manner. Furthermore, 17-AAG administration was shown to induce a pronounced downregulation of multiple Hsp90 protein clients and other downstream effectors, such as IGF-IR, Akt, IKK-α, IKK-β, FOXO1, ERK1/2 and c-Met, resulting in sequestration-mediated inactivation of NF-κB, reduced cell proliferation and decline of cell motility. In total, we have clearly evinced a dose-dependent and cell type-specific effect of 17-AAG on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of multiple Hsp90 clients and subsequent disruption of signaling integrity

  8. MEGADOCK-Web: an integrated database of high-throughput structure-based protein-protein interaction predictions.

    Science.gov (United States)

    Hayashi, Takanori; Matsuzaki, Yuri; Yanagisawa, Keisuke; Ohue, Masahito; Akiyama, Yutaka

    2018-05-08

    Protein-protein interactions (PPIs) play several roles in living cells, and computational PPI prediction is a major focus of many researchers. The three-dimensional (3D) structure and binding surface are important for the design of PPI inhibitors. Therefore, rigid body protein-protein docking calculations for two protein structures are expected to allow elucidation of PPIs different from known complexes in terms of 3D structures because known PPI information is not explicitly required. We have developed rapid PPI prediction software based on protein-protein docking, called MEGADOCK. In order to fully utilize the benefits of computational PPI predictions, it is necessary to construct a comprehensive database to gather prediction results and their predicted 3D complex structures and to make them easily accessible. Although several databases exist that provide predicted PPIs, the previous databases do not contain a sufficient number of entries for the purpose of discovering novel PPIs. In this study, we constructed an integrated database of MEGADOCK PPI predictions, named MEGADOCK-Web. MEGADOCK-Web provides more than 10 times the number of PPI predictions than previous databases and enables users to conduct PPI predictions that cannot be found in conventional PPI prediction databases. In MEGADOCK-Web, there are 7528 protein chains and 28,331,628 predicted PPIs from all possible combinations of those proteins. Each protein structure is annotated with PDB ID, chain ID, UniProt AC, related KEGG pathway IDs, and known PPI pairs. Additionally, MEGADOCK-Web provides four powerful functions: 1) searching precalculated PPI predictions, 2) providing annotations for each predicted protein pair with an experimentally known PPI, 3) visualizing candidates that may interact with the query protein on biochemical pathways, and 4) visualizing predicted complex structures through a 3D molecular viewer. MEGADOCK-Web provides a huge amount of comprehensive PPI predictions based on

  9. Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

    Science.gov (United States)

    Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2018-07-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

  10. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

    Science.gov (United States)

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-10-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

  11. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    Science.gov (United States)

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  12. Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase.

    Science.gov (United States)

    Kwon, Yong-Chan; Oh, In-Seok; Lee, Nahum; Lee, Kyung-Ho; Yoon, Yeo Joon; Lee, Eun Yeol; Kim, Byung-Gee; Kim, Dong-Myung

    2013-04-01

    Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis. Copyright © 2012 Wiley Periodicals, Inc.

  13. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.

    1989-01-01

    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  14. Predicting Protein Function via Semantic Integration of Multiple Networks.

    Science.gov (United States)

    Yu, Guoxian; Fu, Guangyuan; Wang, Jun; Zhu, Hailong

    2016-01-01

    Determining the biological functions of proteins is one of the key challenges in the post-genomic era. The rapidly accumulated large volumes of proteomic and genomic data drives to develop computational models for automatically predicting protein function in large scale. Recent approaches focus on integrating multiple heterogeneous data sources and they often get better results than methods that use single data source alone. In this paper, we investigate how to integrate multiple biological data sources with the biological knowledge, i.e., Gene Ontology (GO), for protein function prediction. We propose a method, called SimNet, to Semantically integrate multiple functional association Networks derived from heterogenous data sources. SimNet firstly utilizes GO annotations of proteins to capture the semantic similarity between proteins and introduces a semantic kernel based on the similarity. Next, SimNet constructs a composite network, obtained as a weighted summation of individual networks, and aligns the network with the kernel to get the weights assigned to individual networks. Then, it applies a network-based classifier on the composite network to predict protein function. Experiment results on heterogenous proteomic data sources of Yeast, Human, Mouse, and Fly show that, SimNet not only achieves better (or comparable) results than other related competitive approaches, but also takes much less time. The Matlab codes of SimNet are available at https://sites.google.com/site/guoxian85/simnet.

  15. Interaction of Proteins Identified in Human Thyroid Cells

    Science.gov (United States)

    Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

    2013-01-01

    Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

  16. Interaction of Proteins Identified in Human Thyroid Cells

    Directory of Open Access Journals (Sweden)

    Jessica Pietsch

    2013-01-01

    Full Text Available Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.

  17. Is Melanoma a stem cell tumor? Identification of neurogenic proteins in trans-differentiated cells

    Directory of Open Access Journals (Sweden)

    Chan Linda S

    2005-03-01

    Full Text Available Abstract Background Although several genes and proteins have been implicated in the development of melanomas, the molecular mechanisms involved in the development of these tumors are not well understood. To gain a better understanding of the relationship between the cell growth, tumorigenesis and differentiation, we have studied a highly malignant cat melanoma cell line that trans-differentiates into neuronal cells after exposure to a feline endogenous retrovirus RD114. Methods To define the repertoire of proteins responsible for the phenotypic differences between melanoma and its counterpart trans-differentiated neuronal cells we have applied proteomics technology and compared protein profiles of the two cell types and identified differentially expressed proteins by 2D-gel electrophoresis, image analyses and mass spectrometry. Results The melanoma and trans-differentiated neuronal cells could be distinguished by the presence of distinct sets of proteins in each. Although approximately 60–70% of the expressed proteins were shared between the two cell types, twelve proteins were induced de novo after infection of melanoma cells with RD114 virus in vitro. Expression of these proteins in trans-differentiated cells was significantly associated with concomitant down regulation of growth promoting proteins and up-regulation of neurogenic proteins (p = 95% proteins expressed in trans-differentiated cells could be associated with the development, differentiation and regulation of nervous system cells. Conclusion Our results indicate that the cat melanoma cells have the ability to differentiate into distinct neuronal cell types and they express proteins that are essential for self-renewal. Since melanocytes arise from the neural crest of the embryo, we conclude that this melanoma arose from embryonic precursor stem cells. This model system provides a unique opportunity to identify domains of interactions between the expressed proteins that halt the

  18. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  19. Whey utilization for single-cell protein production

    Energy Technology Data Exchange (ETDEWEB)

    Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

    1980-01-01

    The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

  20. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  1. A data integration approach for cell cycle analysis oriented to model simulation in systems biology

    Directory of Open Access Journals (Sweden)

    Mosca Ettore

    2007-08-01

    Full Text Available Abstract Background The cell cycle is one of the biological processes most frequently investigated in systems biology studies and it involves the knowledge of a large number of genes and networks of protein interactions. A deep knowledge of the molecular aspect of this biological process can contribute to making cancer research more accurate and innovative. In this context the mathematical modelling of the cell cycle has a relevant role to quantify the behaviour of each component of the systems. The mathematical modelling of a biological process such as the cell cycle allows a systemic description that helps to highlight some features such as emergent properties which could be hidden when the analysis is performed only from a reductionism point of view. Moreover, in modelling complex systems, a complete annotation of all the components is equally important to understand the interaction mechanism inside the network: for this reason data integration of the model components has high relevance in systems biology studies. Description In this work, we present a resource, the Cell Cycle Database, intended to support systems biology analysis on the Cell Cycle process, based on two organisms, yeast and mammalian. The database integrates information about genes and proteins involved in the cell cycle process, stores complete models of the interaction networks and allows the mathematical simulation over time of the quantitative behaviour of each component. To accomplish this task, we developed, a web interface for browsing information related to cell cycle genes, proteins and mathematical models. In this framework, we have implemented a pipeline which allows users to deal with the mathematical part of the models, in order to solve, using different variables, the ordinary differential equation systems that describe the biological process. Conclusion This integrated system is freely available in order to support systems biology research on the cell cycle and

  2. P185-M Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search Engines

    Science.gov (United States)

    Hufnagel, P.; Glandorf, J.; Körting, G.; Jabs, W.; Schweiger-Hufnagel, U.; Hahner, S.; Lubeck, M.; Suckau, D.

    2007-01-01

    Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

  3. System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

    Science.gov (United States)

    Malina, Halina Z

    2011-01-19

    The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules

  4. Systematic analysis of protein turnover in primary cells.

    Science.gov (United States)

    Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

    2018-02-15

    A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

  5. Distribution of DNA replication proteins in Drosophila cells

    Science.gov (United States)

    Easwaran, Hariharan P; Leonhardt, Heinrich; Cardoso, M Cristina

    2007-01-01

    Background DNA replication in higher eukaryotic cells is organized in discrete subnuclear sites called replication foci (RF). During the S phase, most replication proteins assemble at the RF by interacting with PCNA via a PCNA binding domain (PBD). This has been shown to occur for many mammalian replication proteins, but it is not known whether this mechanism is conserved in evolution. Results Fluorescent fusions of mammalian replication proteins, Dnmt1, HsDNA Lig I and HsPCNA were analyzed for their ability to target to RF in Drosophila cells. Except for HsPCNA, none of the other proteins and their deletions showed any accumulation at RF in Drosophila cells. We hypothesized that in Drosophila cells there might be some other peptide sequence responsible for targeting proteins to RF. To test this, we identified the DmDNA Lig I and compared the protein sequence with HsDNA Lig I. The two orthologs shared the PBD suggesting a functionally conserved role for this domain in the Drosophila counterpart. A series of deletions of DmDNA Lig I were analyzed for their ability to accumulate at RF in Drosophila and mammalian cells. Surprisingly, no accumulation at RF was observed in Drosophila cells, while in mammalian cells DmDNA Lig I accumulated at RF via its PBD. Further, GFP fusions with the PBD domains from Dnmt1, HsDNA Lig I and DmDNA Lig I, were able to target to RF only in mammalian cells but not in Drosophila cells. Conclusion We show that S phase in Drosophila cells is characterized by formation of RF marked by PCNA like in mammalian cells. However, other than PCNA none of the replication proteins and their deletions tested here showed accumulation at RF in Drosophila cells while the same proteins and deletions are capable of accumulating at RF in mammalian cells. We hypothesize that unlike mammalian cells, in Drosophila cells, replication proteins do not form long-lasting interactions with the replication machinery, and rather perform their functions via very

  6. Integrating protein structures and precomputed genealogies in the Magnum database: Examples with cellular retinoid binding proteins

    Directory of Open Access Journals (Sweden)

    Bradley Michael E

    2006-02-01

    Full Text Available Abstract Background When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. Results The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1 multiple sequence alignments, 2 mapping of alignment sites to crystal structure sites, 3 phylogenetic trees, 4 inferred ancestral sequences at internal tree nodes, and 5 amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. Conclusion We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural

  7. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak; de Laté , Perle Latré ; Kennedy, Eileen J.; Langsley, Gordon

    2017-01-01

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way

  8. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes.

    Science.gov (United States)

    Ackermann, Amanda M; Wang, Zhiping; Schug, Jonathan; Naji, Ali; Kaestner, Klaus H

    2016-03-01

    Although glucagon-secreting α-cells and insulin-secreting β-cells have opposing functions in regulating plasma glucose levels, the two cell types share a common developmental origin and exhibit overlapping transcriptomes and epigenomes. Notably, destruction of β-cells can stimulate repopulation via transdifferentiation of α-cells, at least in mice, suggesting plasticity between these cell fates. Furthermore, dysfunction of both α- and β-cells contributes to the pathophysiology of type 1 and type 2 diabetes, and β-cell de-differentiation has been proposed to contribute to type 2 diabetes. Our objective was to delineate the molecular properties that maintain islet cell type specification yet allow for cellular plasticity. We hypothesized that correlating cell type-specific transcriptomes with an atlas of open chromatin will identify novel genes and transcriptional regulatory elements such as enhancers involved in α- and β-cell specification and plasticity. We sorted human α- and β-cells and performed the "Assay for Transposase-Accessible Chromatin with high throughput sequencing" (ATAC-seq) and mRNA-seq, followed by integrative analysis to identify cell type-selective gene regulatory regions. We identified numerous transcripts with either α-cell- or β-cell-selective expression and discovered the cell type-selective open chromatin regions that correlate with these gene activation patterns. We confirmed cell type-selective expression on the protein level for two of the top hits from our screen. The "group specific protein" (GC; or vitamin D binding protein) was restricted to α-cells, while CHODL (chondrolectin) immunoreactivity was only present in β-cells. Furthermore, α-cell- and β-cell-selective ATAC-seq peaks were identified to overlap with known binding sites for islet transcription factors, as well as with single nucleotide polymorphisms (SNPs) previously identified as risk loci for type 2 diabetes. We have determined the genetic landscape of

  9. Integrating complex functions: coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins.

    Science.gov (United States)

    Schneiter, Roger; Cole, Charles N

    2010-01-01

    The nuclear envelope harbors numerous large proteinaceous channels, the nuclear pore complexes (NPCs), through which macromolecular exchange between the cytosol and the nucleoplasm occurs. This double-membrane nuclear envelope is continuous with the endoplasmic reticulum and thus functionally connected to such diverse processes as vesicular transport, protein maturation and lipid synthesis. Recent results obtained from studies in Saccharomyces cerevisiae indicate that assembly of the nuclear pore complex is functionally dependent upon maintenance of lipid homeostasis of the ER membrane. Previous work from one of our laboratories has revealed that an integral membrane protein Apq12 is important for the assembly of functional nuclear pores. Cells lacking APQ12 are viable but cannot grow at low temperatures, have aberrant NPCs and a defect in mRNA export. Remarkably, these defects in NPC assembly can be overcome by supplementing cells with a membrane fluidizing agent, benzyl alcohol, suggesting that Apq12 impacts the flexibility of the nuclear membrane, possibly by adjusting its lipid composition when cells are shifted to a reduced temperature. Our new study now expands these findings and reveals that an essential membrane protein, Brr6, shares at least partially overlapping functions with Apq12 and is also required for assembly of functional NPCs. A third nuclear envelope membrane protein, Brl1, is related to Brr6, and is also required for NPC assembly. Because maintenance of membrane homeostasis is essential for cellular survival, the fact that these three proteins are conserved in fungi that undergo closed mitoses, but are not found in metazoans or plants, may indicate that their functions are performed by proteins unrelated at the primary sequence level to Brr6, Brl1 and Apq12 in cells that disassemble their nuclear envelopes during mitosis.

  10. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  11. Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.

    Science.gov (United States)

    Aon, Juan C; Sun, Jianxin; Leighton, Julie M; Appelbaum, Edward R

    2016-08-15

    In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway. The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale. The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.

  12. Enterococcus faecium NCIMB 10415 Modulates Epithelial Integrity, Heat Shock Protein, and Proinflammatory Cytokine Response in Intestinal Cells

    Directory of Open Access Journals (Sweden)

    Shanti Klingspor

    2015-01-01

    Full Text Available Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium, a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2 and human (Caco-2 intestinal cells were incubated without bacteria (control, with E. faecium, with enteropathogenic (EPEC or enterotoxigenic E. coli (ETEC each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.

  13. RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

    Science.gov (United States)

    Galloway, Alison; Saveliev, Alexander; Łukasiak, Sebastian; Hodson, Daniel J; Bolland, Daniel; Balmanno, Kathryn; Ahlfors, Helena; Monzón-Casanova, Elisa; Mannurita, Sara Ciullini; Bell, Lewis S; Andrews, Simon; Díaz-Muñoz, Manuel D; Cook, Simon J; Corcoran, Anne; Turner, Martin

    2016-04-22

    Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint. Copyright © 2016, American Association for the Advancement of Science.

  14. Integrated circuit cell library

    Science.gov (United States)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  15. Engineered mammalian cells for production of recombinant proteins

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

  16. A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis.

    Directory of Open Access Journals (Sweden)

    Michal Shoshkes Carmel

    Full Text Available VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3 proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT. Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos.Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.

  17. Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures

    International Nuclear Information System (INIS)

    Lam, Alan Tin-Lun; Chen, Allen Kuan-Liang; Ting, Sherwin Qi-Peng; Reuveny, Shaul; Oh, Steve Kah-Weng

    2016-01-01

    Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles of such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described. - Highlights: • Expansion of hPSC on microcarriers. • Differentiation of hPSC on microcarriers. • Parameters that can affect the expansion and differentiation of hPSC on microcarriers. • Integration of expansion and differentiation of hPSC on microcarriers in one unit operation.

  18. Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures

    Energy Technology Data Exchange (ETDEWEB)

    Lam, Alan Tin-Lun, E-mail: alan_lam@bti.a-star.edu.sg; Chen, Allen Kuan-Liang; Ting, Sherwin Qi-Peng; Reuveny, Shaul; Oh, Steve Kah-Weng, E-mail: steve_oh@bti.a-star.edu.sg

    2016-05-06

    Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles of such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described. - Highlights: • Expansion of hPSC on microcarriers. • Differentiation of hPSC on microcarriers. • Parameters that can affect the expansion and differentiation of hPSC on microcarriers. • Integration of expansion and differentiation of hPSC on microcarriers in one unit operation.

  19. A biomolecular proportional integral controller based on feedback regulations of protein level and activity.

    Science.gov (United States)

    Mairet, Francis

    2018-02-01

    Homeostasis is the capacity of living organisms to keep internal conditions regulated at a constant level, despite environmental fluctuations. Integral feedback control is known to play a key role in this behaviour. Here, I show that a feedback system involving transcriptional and post-translational regulations of the same executor protein acts as a proportional integral (PI) controller, leading to enhanced transient performances in comparison with a classical integral loop. Such a biomolecular controller-which I call a level and activity-PI controller (LA-PI)-is involved in the regulation of ammonium uptake by Escherichia coli through the transporter AmtB. The P II molecules, which reflect the nitrogen status of the cell, inhibit both the production of AmtB and its activity (via the NtrB-NtrC system and the formation of a complex with GlnK, respectively). Other examples of LA-PI controller include copper and zinc transporters, and the redox regulation in photosynthesis. This scheme has thus emerged through evolution in many biological systems, surely because of the benefits it offers in terms of performances (rapid and perfect adaptation) and economy (protein production according to needs).

  20. Use of non-conventional cell disruption method for extraction of proteins from black yeasts

    Directory of Open Access Journals (Sweden)

    Maja eLeitgeb

    2016-04-01

    Full Text Available The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2 by varying pressure at fixed temperature (35 °C. The black yeasts cell walls were disrupted and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV-Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2 treated cells. The advantages of the proposed method are in a simple use which is also possible for heat sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions.

  1. Use of Non-Conventional Cell Disruption Method for Extraction of Proteins from Black Yeasts

    Science.gov (United States)

    Čolnik, Maja; Primožič, Mateja; Knez, Željko; Leitgeb, Maja

    2016-01-01

    The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions. PMID:27148527

  2. Usher protein functions in hair cells and photoreceptors.

    Science.gov (United States)

    Cosgrove, Dominic; Zallocchi, Marisa

    2014-01-01

    The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  4. Fluorescent tags of protein function in living cells.

    Science.gov (United States)

    Whitaker, M

    2000-02-01

    A cell's biochemistry is now known to be the biochemistry of molecular machines, that is, protein complexes that are assembled and dismantled in particular locations within the cell as needed. One important element in our understanding has been the ability to begin to see where proteins are in cells and what they are doing as they go about their business. Accordingly, there is now a strong impetus to discover new ways of looking at the workings of proteins in living cells. Although the use of fluorescent tags to track individual proteins in cells has a long history, the availability of laser-based confocal microscopes and the imaginative exploitation of the green fluorescent protein from jellyfish have provided new tools of great diversity and utility. It is now possible to watch a protein bind its substrate or its partners in real time and with submicron resolution within a single cell. The importance of processes of self-organisation represented by protein folding on the one hand and subcellular organelles on the other are well recognised. Self-organisation at the intermediate level of multimeric protein complexes is now open to inspection. BioEssays 22:180-187, 2000. Copyright 2000 John Wiley & Sons, Inc.

  5. Outside-in control -Does plant cell wall integrity regulate cell cycle progression?

    Science.gov (United States)

    Gigli-Bisceglia, Nora; Hamann, Thorsten

    2018-04-13

    During recent years it has become accepted that plant cell walls are not inert objects surrounding all plant cells but are instead highly dynamic, plastic structures. They are involved in a large number of cell biological processes and contribute actively to plant growth, development and interaction with environment. Therefore, it is not surprising that cellular processes can control plant cell wall integrity while, simultaneously, cell wall integrity can influence cellular processes. In yeast and animal cells such a bi-directional relationship also exists between the yeast/animal extra-cellular matrices and the cell cycle. In yeast, the cell wall integrity maintenance mechanism and a dedicated plasmamembrane integrity checkpoint are mediating this relationship. Recent research has yielded insights into the mechanism controlling plant cell wall metabolism during cytokinesis. However, knowledge regarding putative regulatory pathways controlling adaptive modifications in plant cell cycle activity in response to changes in the state of the plant cell wall are not yet identified. In this review, we summarize similarities and differences in regulatory mechanisms coordinating extra cellular matrices and cell cycle activity in animal and yeast cells, discuss the available evidence supporting the existence of such a mechanism in plants and suggest that the plant cell wall integrity maintenance mechanism might also control cell cycle activity in plant cells. This article is protected by copyright. All rights reserved.

  6. High throughput platforms for structural genomics of integral membrane proteins.

    Science.gov (United States)

    Mancia, Filippo; Love, James

    2011-08-01

    Structural genomics approaches on integral membrane proteins have been postulated for over a decade, yet specific efforts are lagging years behind their soluble counterparts. Indeed, high throughput methodologies for production and characterization of prokaryotic integral membrane proteins are only now emerging, while large-scale efforts for eukaryotic ones are still in their infancy. Presented here is a review of recent literature on actively ongoing structural genomics of membrane protein initiatives, with a focus on those aimed at implementing interesting techniques aimed at increasing our rate of success for this class of macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  8. Thermodynamics of protein destabilization in live cells.

    Science.gov (United States)

    Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

    2015-10-06

    Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

  9. Cell-free protein synthesis: applications in proteomics and biotechnology.

    Science.gov (United States)

    He, Mingyue

    2008-01-01

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

  10. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas.

    Directory of Open Access Journals (Sweden)

    Priscila Daniele Ramos Cirilo

    Full Text Available Uterine Leiomyomas (ULs are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs. Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs.We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC and gene expression microarrays (SAM. The CONEXIC algorithm was applied to integrate the data.The integrated analysis identified the top 30 significant genes (P<0.01, which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively and IGFBP5 (P = 0.0002 and P = 0.006, respectively were up-regulated in the tumours when compared with the adjacent normal myometrium.The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.

  11. H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

    KAUST Repository

    Zolea, Fabiana

    2015-10-09

    The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

  12. H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

    KAUST Repository

    Zolea, Fabiana; Biamonte, Flavia; Candeloro, Patrizio; Di Sanzo, Maddalena; Cozzi, Anna; Di Vito, Anna; Quaresima, Barbara; Lobello, Nadia; Trecroci, Francesca; Di Fabrizio, Enzo M.; Levi, Sonia; Cuda, Giovanni; Costanzo, Francesco

    2015-01-01

    The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

  13. Integrative omics analysis. A study based on Plasmodium falciparum mRNA and protein data.

    Science.gov (United States)

    Tomescu, Oana A; Mattanovich, Diethard; Thallinger, Gerhard G

    2014-01-01

    Technological improvements have shifted the focus from data generation to data analysis. The availability of large amounts of data from transcriptomics, protemics and metabolomics experiments raise new questions concerning suitable integrative analysis methods. We compare three integrative analysis techniques (co-inertia analysis, generalized singular value decomposition and integrative biclustering) by applying them to gene and protein abundance data from the six life cycle stages of Plasmodium falciparum. Co-inertia analysis is an analysis method used to visualize and explore gene and protein data. The generalized singular value decomposition has shown its potential in the analysis of two transcriptome data sets. Integrative Biclustering applies biclustering to gene and protein data. Using CIA, we visualize the six life cycle stages of Plasmodium falciparum, as well as GO terms in a 2D plane and interpret the spatial configuration. With GSVD, we decompose the transcriptomic and proteomic data sets into matrices with biologically meaningful interpretations and explore the processes captured by the data sets. IBC identifies groups of genes, proteins, GO Terms and life cycle stages of Plasmodium falciparum. We show method-specific results as well as a network view of the life cycle stages based on the results common to all three methods. Additionally, by combining the results of the three methods, we create a three-fold validated network of life cycle stage specific GO terms: Sporozoites are associated with transcription and transport; merozoites with entry into host cell as well as biosynthetic and metabolic processes; rings with oxidation-reduction processes; trophozoites with glycolysis and energy production; schizonts with antigenic variation and immune response; gametocyctes with DNA packaging and mitochondrial transport. Furthermore, the network connectivity underlines the separation of the intraerythrocytic cycle from the gametocyte and sporozoite stages

  14. Role for protein geranylgeranylation in adult T-cell leukemia cell survival

    International Nuclear Information System (INIS)

    Nonaka, Mizuho; Uota, Shin; Saitoh, Yasunori; Takahashi, Mayumi; Sugimoto, Haruyo; Amet, Tohti; Arai, Ayako; Miura, Osamu; Yamamoto, Naoki; Yamaoka, Shoji

    2009-01-01

    Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL

  15. Biosynthesis and release of proteins by isolated pulmonary Clara cells

    International Nuclear Information System (INIS)

    Patton, S.E.; Gilmore, L.B.; Jetten, A.M.; Nettesheim, P.; Hook, G.E.

    1986-01-01

    The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35 S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14 C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14 C-glucosamine indicating that it was not a glycoprotein. Data demonstrate the release of several proteins from isolated Clara cells but the major protein had a M.W. of 6 Kd

  16. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

  17. Human Cells as Platform to Produce Gamma-Carboxylated Proteins.

    Science.gov (United States)

    de Sousa Bomfim, Aline; de Freitas, Marcela Cristina Corrêa; Covas, Dimas Tadeu; de Sousa Russo, Elisa Maria

    2018-01-01

    The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.

  18. Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

    Science.gov (United States)

    Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

    1978-09-01

    Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

  19. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    Science.gov (United States)

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  20. Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity.

    Directory of Open Access Journals (Sweden)

    Antoine de Morrée

    Full Text Available Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.

  1. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    International Nuclear Information System (INIS)

    Zhang, Heyu; Ma, Xi; Shi, Taiping; Song, Quansheng; Zhao, Hongshan; Ma, Dalong

    2010-01-01

    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  2. CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.

    Science.gov (United States)

    Baillat, David; Russell, William K; Wagner, Eric J

    2016-12-01

    The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  4. The Craterostigma plantagineum glycine-rich protein CpGRP1 interacts with a cell wall-associated protein kinase 1 (CpWAK1) and accumulates in leaf cell walls during dehydration.

    Science.gov (United States)

    Giarola, Valentino; Krey, Stephanie; von den Driesch, Barbara; Bartels, Dorothea

    2016-04-01

    Craterostigma plantagineum tolerates extreme desiccation. Leaves of this plant shrink and extensively fold during dehydration and expand again during rehydration, preserving their structural integrity. Genes were analysed that may participate in the reversible folding mechanism. Analysis of transcripts abundantly expressed in desiccated leaves identified a gene putatively coding for an apoplastic glycine-rich protein (CpGRP1). We studied the expression, regulation and subcellular localization of CpGRP1 and its ability to interact with a cell wall-associated protein kinase (CpWAK1) to understand the role of CpGRP1 in the cell wall during dehydration. The CpGRP1 protein accumulates in the apoplast of desiccated leaves. Analysis of the promoter revealed that the gene expression is mainly regulated at the transcriptional level, is independent of abscisic acid (ABA) and involves a drought-responsive cis-element (DRE). CpGRP1 interacts with CpWAK1 which is down-regulated in response to dehydration. Our data suggest a role of the CpGRP1-CpWAK1 complex in dehydration-induced morphological changes in the cell wall during dehydration in C. plantagineum. Cell wall pectins and dehydration-induced pectin modifications are predicted to be involved in the activity of the CpGRP1-CpWAK1 complex. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  5. Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

    Science.gov (United States)

    Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

    2018-05-18

    The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

  6. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    Science.gov (United States)

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  7. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

    Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  8. Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stem cells

    OpenAIRE

    Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S.; Woollard, John R.; Tang, Hui; Dasari, Surendra; Lerman, Amir; van Wijnen, Andre J.; Lerman, Lilach O.

    2017-01-01

    Background Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs. Methods Simultaneous expression profiles of m...

  9. HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma—A Tale of Two Proteins: Tax and HBZ

    Directory of Open Access Journals (Sweden)

    Chou-Zen Giam

    2016-06-01

    Full Text Available HTLV-1 (Human T-cell lymphotropic virus type 1 is a complex human delta retrovirus that currently infects 10–20 million people worldwide. While HTLV-1 infection is generally asymptomatic, 3%–5% of infected individuals develop a highly malignant and intractable T-cell neoplasm known as adult T-cell leukemia/lymphoma (ATL decades after infection. How HTLV-1 infection progresses to ATL is not well understood. Two viral regulatory proteins, Tax and HTLV-1 basic zipper protein (HBZ, encoded by the sense and antisense viral transcripts, respectively, are thought to play indispensable roles in the oncogenic process of ATL. This review focuses on the roles of Tax and HBZ in viral replication, persistence, and oncogenesis. Special emphasis is directed towards recent literature on the mechanisms of action of these two proteins and the roles of Tax and HBZ in influencing the outcomes of HTLV-1 infection including senescence induction, viral latency and persistence, genome instability, cell proliferation, and ATL development. Attempts are made to integrate results from cell-based studies of HTLV-1 infection and studies of HTLV-1 proviral integration site preference, clonality, and clonal expansion based on high throughput DNA sequencing. Recent data showing that Tax hijacks key mediators of DNA double-strand break repair signaling—the ubiquitin E3 ligase, ring finger protein 8 (RNF8 and the ubiquitin E2 conjugating enzyme (UBC13—to activate the canonical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB and other signaling pathways will be discussed. A perspective on how the Tax-RNF8 signaling axis might impact genomic instability and how Tax may collaborate with HBZ to drive oncogenesis is provided.

  10. HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma—A Tale of Two Proteins: Tax and HBZ

    Science.gov (United States)

    Giam, Chou-Zen; Semmes, Oliver John

    2016-01-01

    HTLV-1 (Human T-cell lymphotropic virus type 1) is a complex human delta retrovirus that currently infects 10–20 million people worldwide. While HTLV-1 infection is generally asymptomatic, 3%–5% of infected individuals develop a highly malignant and intractable T-cell neoplasm known as adult T-cell leukemia/lymphoma (ATL) decades after infection. How HTLV-1 infection progresses to ATL is not well understood. Two viral regulatory proteins, Tax and HTLV-1 basic zipper protein (HBZ), encoded by the sense and antisense viral transcripts, respectively, are thought to play indispensable roles in the oncogenic process of ATL. This review focuses on the roles of Tax and HBZ in viral replication, persistence, and oncogenesis. Special emphasis is directed towards recent literature on the mechanisms of action of these two proteins and the roles of Tax and HBZ in influencing the outcomes of HTLV-1 infection including senescence induction, viral latency and persistence, genome instability, cell proliferation, and ATL development. Attempts are made to integrate results from cell-based studies of HTLV-1 infection and studies of HTLV-1 proviral integration site preference, clonality, and clonal expansion based on high throughput DNA sequencing. Recent data showing that Tax hijacks key mediators of DNA double-strand break repair signaling—the ubiquitin E3 ligase, ring finger protein 8 (RNF8) and the ubiquitin E2 conjugating enzyme (UBC13)—to activate the canonical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and other signaling pathways will be discussed. A perspective on how the Tax-RNF8 signaling axis might impact genomic instability and how Tax may collaborate with HBZ to drive oncogenesis is provided. PMID:27322308

  11. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  12. Femtosecond UV-laser pulses to unveil protein-protein interactions in living cells.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria M; Della Ventura, Bartolomeo; Vinciguerra, Roberto; Chino, Marco; Gesuele, Felice; Lombardi, Angelina; Velotta, Raffaele; Altucci, Carlo; Birolo, Leila; Piccoli, Renata; Arciello, Angela

    2016-02-01

    A hallmark to decipher bioprocesses is to characterize protein-protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

  13. Nanoparticles-cell association predicted by protein corona fingerprints

    Science.gov (United States)

    Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

    2016-06-01

    In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface

  14. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  15. Production of membrane proteins without cells or detergents.

    Science.gov (United States)

    Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

    2011-04-30

    The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. The Integration of Bacteriorhodopsin Proteins with Semiconductor Heterostructure Devices

    Science.gov (United States)

    Xu, Jian

    2008-03-01

    Bioelectronics has emerged as one of the most rapidly developing fields among the active frontiers of interdisciplinary research. A major thrust in this field is aimed at the coupling of the technologically-unmatched performance of biological systems, such as neural and sensing functions, with the well developed technology of microelectronics and optoelectronics. To this end we have studied the integration of a suitably engineered protein, bacteriorhodopsin (BR), with semiconductor optoelectronic devices and circuits. Successful integration will potentially lead to ultrasensitive sensors with polarization selectivity and built-in preprocessing capabilities that will be useful for high speed tracking, motion and edge detection, biological detection, and artificial vision systems. In this presentation we will summarize our progresses in this area, which include fundamental studies on the transient dynamics of photo-induced charge shift in BR and the coupling mechanism at protein-semiconductor interface for effective immobilizing and selectively integrating light sensitive proteins with microelectronic devices and circuits, and the device engineering of BR-transistor-integrated optical sensors as well as their applications in phototransceiver circuits. Work done in collaboration with Pallab Bhattacharya, Jonghyun Shin, Department of Electrical Engineering and Computer Science, University of Michigan, Ann Arbor, MI; Robert R. Birge, Department of Chemistry, University of Connecticut, Storrs, CT 06269; and György V'ar'o, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Science, H-6701 Szeged, Hungary.

  17. Tissue engineering and cell-based therapy toward integrated strategy with artificial organs.

    Science.gov (United States)

    Gojo, Satoshi; Toyoda, Masashi; Umezawa, Akihiro

    2011-09-01

    Research in order that artificial organs can supplement or completely replace the functions of impaired or damaged tissues and internal organs has been underway for many years. The recent clinical development of implantable left ventricular assist devices has revolutionized the treatment of patients with heart failure. The emerging field of regenerative medicine, which uses human cells and tissues to regenerate internal organs, is now advancing from basic and clinical research to clinical application. In this review, we focus on the novel biomaterials, i.e., fusion protein, and approaches such as three-dimensional and whole-organ tissue engineering. We also compare induced pluripotent stem cells, directly reprogrammed cardiomyocytes, and somatic stem cells for cell source of future cell-based therapy. Integrated strategy of artificial organ and tissue engineering/regenerative medicine should give rise to a new era of medical treatment to organ failure.

  18. Methods for production of proteins in host cells

    Science.gov (United States)

    Donnelly, Mark; Joachimiak, Andrzej

    2004-01-13

    The present invention provides methods for the production of proteins, particularly toxic proteins, in host cells. The invention provides methods which use a fusion protein comprising a chaperonin binding domain in host cells induced or regulated to have increased levels of chaperonin which binds the chaperonin binding domain.

  19. Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

  20. Short-term effects of ultrahigh concentration cationic silica nanoparticles on cell internalization, cytotoxicity, and cell integrity with human breast cancer cell line (MCF-7)

    Energy Technology Data Exchange (ETDEWEB)

    Seog, Ji Hyun [Korea Advanced Institute of Science and Technology, Graduate School of Nanoscience and Technology (Korea, Republic of); Kong, Bokyung [Corning Precision Materials (Korea, Republic of); Kim, Dongheun [Korea Advanced Institute of Science and Technology, Graduate School of Nanoscience and Technology (Korea, Republic of); Graham, Lauren M. [University of Maryland, Department of Chemistry and Biochemistry (United States); Choi, Joon Sig [Chungnam National University, Department of Biochemistry (Korea, Republic of); Lee, Sang Bok, E-mail: slee@umd.edu [Korea Advanced Institute of Science and Technology, Graduate School of Nanoscience and Technology (Korea, Republic of)

    2015-01-15

    High concentrations of cationic colloidal silica nanoparticles (CCS-NPs) have been widely used for the enrichment of plasma membrane proteins. However, the interaction between the CCS-NPs and cells under the required concentration for the isolation of plasma membrane are rarely investigated. We evaluated the internalization and toxicity of the 15 nm CCS-NPs which were exposed at high concentrations with short time in human breast cancer cells (MCF-7) with transmission electron microscopy, energy dispersive X-ray spectroscopy, inductively coupled plasma atomic emission spectroscopy, and colorimetric assays. The NPs were observed throughout the cells, particularly in the cytoplasm and the nucleus, after short incubation periods. Additionally, the NPs significantly influenced the membrane integrity of the MCF-7 cells.

  1. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    Science.gov (United States)

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  2. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host-Cell Interaction.

    Science.gov (United States)

    Gil-Bona, Ana; Reales-Calderon, Jose A; Parra-Giraldo, Claudia M; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a "veil growth," never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

  3. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

    Science.gov (United States)

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2016-01-12

    Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells.

    Science.gov (United States)

    Cunha, Bárbara; Aguiar, Tiago; Silva, Marta M; Silva, Ricardo J S; Sousa, Marcos F Q; Pineda, Earl; Peixoto, Cristina; Carrondo, Manuel J T; Serra, Margarida; Alves, Paula M

    2015-11-10

    The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover, we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells, with high cell recovery (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, an integrated process allowed for a shorter process time, recovering 70% of viable hMSC (>95%), with no changes in terms of morphology, immunophenotype, proliferation capacity and multipotent differentiation potential. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

    Science.gov (United States)

    Tong, X; Kono, T; Evans-Molina, C

    2015-06-18

    The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

  6. Mining disease genes using integrated protein-protein interaction and gene-gene co-regulation information.

    Science.gov (United States)

    Li, Jin; Wang, Limei; Guo, Maozu; Zhang, Ruijie; Dai, Qiguo; Liu, Xiaoyan; Wang, Chunyu; Teng, Zhixia; Xuan, Ping; Zhang, Mingming

    2015-01-01

    In humans, despite the rapid increase in disease-associated gene discovery, a large proportion of disease-associated genes are still unknown. Many network-based approaches have been used to prioritize disease genes. Many networks, such as the protein-protein interaction (PPI), KEGG, and gene co-expression networks, have been used. Expression quantitative trait loci (eQTLs) have been successfully applied for the determination of genes associated with several diseases. In this study, we constructed an eQTL-based gene-gene co-regulation network (GGCRN) and used it to mine for disease genes. We adopted the random walk with restart (RWR) algorithm to mine for genes associated with Alzheimer disease. Compared to the Human Protein Reference Database (HPRD) PPI network alone, the integrated HPRD PPI and GGCRN networks provided faster convergence and revealed new disease-related genes. Therefore, using the RWR algorithm for integrated PPI and GGCRN is an effective method for disease-associated gene mining.

  7. Protein Availability and Satellite Cell Dynamics in Skeletal Muscle.

    Science.gov (United States)

    Shamim, Baubak; Hawley, John A; Camera, Donny M

    2018-06-01

    Human skeletal muscle satellite cells are activated in response to both resistance and endurance exercise. It was initially proposed that satellite cell proliferation and differentiation were only required to support resistance exercise-induced hypertrophy. However, satellite cells may also play a role in muscle fibre remodelling after endurance-based exercise and extracellular matrix regulation. Given the importance of dietary protein, particularly branched chain amino acids, in supporting myofibrillar and mitochondrial adaptations to both resistance and endurance-based training, a greater understanding of how protein intake impacts satellite cell activity would provide further insight into the mechanisms governing skeletal muscle remodelling with exercise. While many studies have investigated the capacity for protein ingestion to increase post-exercise rates of muscle protein synthesis, few investigations have examined the role for protein ingestion to modulate satellite cell activity. Here we review the molecular mechanisms controlling the activation of satellite cells in response to mechanical stress and protein intake in both in vitro and in vivo models. We provide a mechanistic framework that describes how protein ingestion may enhance satellite activity and promote exercise adaptations in human skeletal muscle.

  8. HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

    Science.gov (United States)

    Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

    2014-01-01

    BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

  9. Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

    Science.gov (United States)

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2018-02-10

    A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  11. Insect Cells as Hosts for Recombinat Proteins

    OpenAIRE

    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  12. Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin.

    Science.gov (United States)

    Udani, M; Zen, Q; Cottman, M; Leonard, N; Jefferson, S; Daymont, C; Truskey, G; Telen, M J

    1998-01-01

    Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors. PMID:9616226

  13. [Non-invasive analysis of proteins in living cells using NMR spectroscopy].

    Science.gov (United States)

    Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

    2015-01-01

    NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

  14. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  15. Effects of hepatitis B virus S protein exposure on sperm membrane integrity and functions.

    Directory of Open Access Journals (Sweden)

    XiangJin Kang

    Full Text Available BACKGROUND: Hepatitis B is a public health problem worldwide. Viral infection can affect a man's fertility, but only scant information about the influence of hepatitis B virus (HBV infection on sperm quality is available. The purpose of this study was to investigate the effect of hepatitis B virus S protein (HBs on human sperm membrane integrity and functions. METHODS/PRINCIPAL FINDINGS: Reactive oxygen species (ROS, lipid peroxidation (LP, total antioxidant capacity (TAC and phosphatidylserine (PS externalization were determined. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assays and flow cytometric analyses were performed. (1 After 3 h incubation with 25 µg/ml of HBs, the average rates of ROS positive cells, annexin V-positive/propidium iodide (PI-negative cells, Caspases-3,-8,-9 positive cells and TUNEL-positive cells were significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. The level of malondialdehyde (MDA in the sperm cells exposed to 50 µg/ml of HBs for 3 h was significantly higher than that in the control (P<0.05-0.01. (2 HBs increased the MDA levels and the numbers of ROS positive cells, annexin V-positive/PI-negative cells, caspases-3, -8, -9 positive cells and TUNEL-positive cells in a dose-dependent manner. (3 HBs monoclonal antibody (MAb and N-Acetylcysteine (NAC reduced the number of ROS-positive sperm cells. (4 HBs decreased the TAC levels in sperm cells in a dose-dependent manner. CONCLUSION: HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, PS externalization, activation of caspases, and DNA fragmentation, resulting in increased apoptosis of sperm cells and loss of sperm membrane integrity and causing sperm dysfunctions.

  16. Congenital heart disease protein 5 associates with CASZ1 to maintain myocardial tissue integrity.

    Science.gov (United States)

    Sojka, Stephen; Amin, Nirav M; Gibbs, Devin; Christine, Kathleen S; Charpentier, Marta S; Conlon, Frank L

    2014-08-01

    The identification and characterization of the cellular and molecular pathways involved in the differentiation and morphogenesis of specific cell types of the developing heart are crucial to understanding the process of cardiac development and the pathology associated with human congenital heart disease. Here, we show that the cardiac transcription factor CASTOR (CASZ1) directly interacts with congenital heart disease 5 protein (CHD5), which is also known as tryptophan-rich basic protein (WRB), a gene located on chromosome 21 in the proposed region responsible for congenital heart disease in individuals with Down's syndrome. We demonstrate that loss of CHD5 in Xenopus leads to compromised myocardial integrity, improper deposition of basement membrane, and a resultant failure of hearts to undergo cell movements associated with cardiac formation. We further report that CHD5 is essential for CASZ1 function and that the CHD5-CASZ1 interaction is necessary for cardiac morphogenesis. Collectively, these results establish a role for CHD5 and CASZ1 in the early stages of vertebrate cardiac development. © 2014. Published by The Company of Biologists Ltd.

  17. Human neuronal cell protein responses to Nipah virus infection

    Directory of Open Access Journals (Sweden)

    Hassan Sharifah

    2007-06-01

    Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

  18. ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases

    OpenAIRE

    Kline, C. Leah B.; Van den Heuvel, A. Pieter J.; Allen, Joshua E.; Prabhu, Varun V.; Dicker, David T.; El-Deiry, Wafik S.

    2016-01-01

    ONC201 (also called TIC10) is a small molecule that inactivates the cell proliferation- and cell survival-promoting kinases AKT and ERK and induces cell death through the pro-apoptotic protein TRAIL. ONC201 is currently in early phase clinical testing for various malignancies. Here, we found through gene expression and protein analyses that ONC201 triggered an increase in TRAIL abundance and cell death through an integrated stress response (ISR) involving the transcription factor ATF4, the tr...

  19. ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

    OpenAIRE

    Grifoni, Samira C.; Jernigan, Nikki L.; Hamilton, Gina; Drummond, Heather A.

    2007-01-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated Epithelial Na+ Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration, however the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence indi...

  20. Protein-Level Integration Strategy of Multiengine MS Spectra Search Results for Higher Confidence and Sequence Coverage.

    Science.gov (United States)

    Zhao, Panpan; Zhong, Jiayong; Liu, Wanting; Zhao, Jing; Zhang, Gong

    2017-12-01

    Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20. As expected, the union of seven engines resulted in a boosted false identification, whereas the intersection of seven engines remarkably decreased the identification power. We found that identifications of at least two out of seven engines resulted in maximizing the protein identification power while minimizing the ratio of suspicious/translation-supported identifications (STR), as monitored by our STR index, based on RNC-Seq. Furthermore, this strategy also significantly improves the peptides coverage of the protein amino acid sequence. In summary, we demonstrated a simple strategy to significantly improve the performance for shotgun mass spectrometry by protein-level integrating multiple search engines, maximizing the utilization of the current MS spectra without additional experimental work.

  1. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    International Nuclear Information System (INIS)

    Takada, Shinako; Koike, Katsuro

    1990-01-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3' end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product

  3. Protein biosynthesis in cultured human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1980-10-31

    A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.

  4. An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

    Czech Academy of Sciences Publication Activity Database

    Hronová, Vladislava; Valášek, Leoš

    2017-01-01

    Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 7.725, year: 2016

  5. The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

    Science.gov (United States)

    Morsczeck, Christian; Hullmann, Markus; Reck, Anja; Reichert, Torsten E

    2018-02-01

    Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of β-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

  6. Role of heat shock proteins in cell apoptosis

    Directory of Open Access Journals (Sweden)

    Arleta Kaźmierczuk

    2010-06-01

    Full Text Available Apoptosis is, apart from necrosis and autophagy, one of the possible cell death mechanisms eliminating needless, not normal or infected cells. This process ensures quantitative and qualitative cell control of organisms. Apoptosis is tightly regulated, it requires both activation of a large number of genes and energy input. Up-to-date two main apoptotic pathways have been recognized – external/receptor and internal, processed with the participation of mitochondria. Heat shock proteins HSPs, the molecules known from their chaperone activity and molecular conservatism, play essential functions in the course of apoptosis. Among that proteins family, i.e. HSP100, 90, 70, 60, 40 and small molecular (sHSP, there are agents mainly protective against programmed cell death. However, in some conditions some of these proteins may promote apoptosis. This review describes different key apoptotic proteins interacting with main members of HSP family and the consequence of these events for cell survival or apoptosis.

  7. ProFASTA: a pipeline web server for fungal protein scanning with integration of cell surface prediction software

    NARCIS (Netherlands)

    de Groot, P.W.J.; Brandt, B.W.

    2012-01-01

    Surface proteins, such as those located in the cell wall of fungi, play an important role in the interaction with the surrounding environment. For instance, they mediate primary host-pathogen interactions and are crucial to the establishment of biofilms and fungal infections. Surface localization of

  8. Production of a soluble recombinant prion protein fused to blue fluorescent protein without refolding or detergents in Escherichia coli cells.

    Science.gov (United States)

    Arii, Yasuhiro; Yamaguchi, Hidenori; Fukuoka, Shin-Ichi

    2007-10-01

    The physiological function of prion proteins (PrP) remains unclear. To investigate the physiological relevance of PrP, we constructed a fusion protein of PrP with enhanced blue fluorescent protein (PrP-EBFP) to quantify the interaction of PrP with other molecules. Production of soluble PrP-EBFP was achieved by lowering the expression temperature in Escherichia coli (E. coli) cells to 15 degrees C. Soluble PrP-EBFP was purified on cation exchange and heparin-affinity columns to yield high purity protein. This is the first report of the preparation of soluble recombinant PrP without refolding following solubilization using denaturants or disruption using detergents. To confirm the integrity of PrP-EBFP, anisotropy was estimated under physiological conditions in the presence of heparin, which interacts with PrP. The dissociation constant was determined to be 0.88+/-0.07 microM. PrP-EBFP should be useful in the quantification of PrP interactions with other molecules.

  9. Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

    International Nuclear Information System (INIS)

    Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

    1988-01-01

    A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

  10. Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

    Science.gov (United States)

    Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

    2013-08-20

    Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus.

    Directory of Open Access Journals (Sweden)

    Mengying Li

    Full Text Available N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth.

  12. Purification and characterization of a soybean cell wall protein

    International Nuclear Information System (INIS)

    San Francisco, S.; Tierney, M.L.

    1989-01-01

    Plant cell wall composition is thought to reflect cellular responses to developmental and environmental signals. We have purified a 33 kDa protein from cell wall extracts of soybean seedlings which is most abundant in extracts from the hook region of the hypocotyl and is rich in proline and hydroxypyroline. In vivo 3 H-proline labelling of hypocotyl tissues indicates that the hook tissue is the predominant site for synthesis of this protein. In unwounded hook, label is incorporated into a 33 kDa protein, while in wounded hook this and additional proteins rich in proline are synthesized. Similarly treated cell wall extracts analyzed by Western blot analysis, using a polyclonal antibody raised against this 33kD protein, showed that the 33 kDa protein is most abundant in cell wall extracts from the hook region of unwounded seedlings and does not increase upon wounding. An immunologically related 35kD protein is also apparent in extracts from wounded hooks and appears to co-migrate with one of the labelled proteins extractable from this tissue. These data indicate that there are two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one of which (33 kDa) is prominent during seedling development and another (35 kDa) which is wound inducible

  13. Integrated fuel cell stack shunt current prevention arrangement

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Robert P. (Cheshire, CT); Nowak, Michael P. (Bolton, CT)

    1992-01-01

    A fuel cell stack includes a plurality of fuel cells juxtaposed with one another in the stack and each including a pair of plate-shaped anode and cathode electrodes that face one another, and a quantity of liquid electrolyte present at least between the electrodes. A separator plate is interposed between each two successive electrodes of adjacent ones of the fuel cells and is unified therewith into an integral separator plate. Each integral separator plate is provided with a circumferentially complete barrier that prevents flow of shunt currents onto and on an outer peripheral surface of the separator plate. This barrier consists of electrolyte-nonwettable barrier members that are accommodated, prior to the formation of the integral separator plate, in corresponding edge recesses situated at the interfaces between the electrodes and the separator plate proper. Each barrier member extends over the entire length of the associated marginal portion and is flush with the outer periphery of the integral separator plate. This barrier also prevents cell-to-cell migration of any electrolyte that may be present at the outer periphery of the integral separator plate while the latter is incorporated in the fuel cell stack.

  14. Singlet oxygen-mediated formation of protein peroxides within cells

    International Nuclear Information System (INIS)

    Wright, A.; Policarpio, V.

    2003-01-01

    Full text: Singlet oxygen is generated by a number of cellular, enzymatic and chemical reactions as well as by exposure to UV, or visible light in the presence of a sensitizer; as a consequence this oxidant has been proposed as a damaging agent in a number of pathologies including photo-aging and skin cancer. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study it is shown that illumination of viable, sensitizer-loaded, THP-1 (human monocyte-like) cells with visible light gives rise to intra-cellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of the sensitizer, is enhanced in D 2 O, and decreased by azide; these data are consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to ca. 1.5 nmoles peroxide per 10 6 cells using rose bengal as sensitizer, or 10 nmoles per mg cell protein and account for up to ca. 15% of the O 2 consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, the cellular protein peroxide levels decreases with t 1/2 ca. 4 hrs at 37 deg C, and this is associated with increased cell lysis. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions, gives rise to radicals as detected by EPR spin trapping. These protein peroxides, and radicals derived from them, can inactivate key cellular enzymes (including caspases, GAPDH and glutathione reductase) and induce DNA base oxidation, strand breaks and DNA-protein cross-links. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer gives rise to novel long-lived, but reactive, intra-cellular protein peroxides via singlet oxygen

  15. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host–Cell Interaction

    Science.gov (United States)

    Gil-Bona, Ana; Reales-Calderon, Jose A.; Parra-Giraldo, Claudia M.; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

  16. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Directory of Open Access Journals (Sweden)

    Sae Tanaka

    Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  17. Polycation-mediated integrated cell death processes

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Andersen, Helene; Wu, Linping

    2014-01-01

    standard. PEIs are highly efficient transfectants, but depending on their architecture and size they induce cytotoxicity through different modes of cell death pathways. Here, we briefly review dynamic and integrated cell death processes and pathways, and discuss considerations in cell death assay design...

  18. Multiple chromosomal gene integration for production of pharmaceutical proteins in S. cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Malene; Mortensen, Uffe Hasbro; Gunnarsson, Nina

    2014-01-01

    When studying protein folding and secretion the general conception is that all cells in a population express an equal amount of protein. Recent work has shown that expression levels vary greatly in cell populations which express proteins on plasmids. Hence a yeast expression platform has been dev...

  19. Integration of AI-2 Based Cell-Cell Signaling with Metabolic Cues in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arindam Mitra

    Full Text Available The quorum sensing molecule Autoinducer-2 (AI-2 is generated as a byproduct of activated methyl cycle by the action of LuxS in Escherichia coli. AI-2 is synthesized, released and later internalized in a cell-density dependent manner. Here, by mutational analysis of the genes, uvrY and csrA, we describe a regulatory circuit of accumulation and uptake of AI-2. We constructed a single-copy chromosomal luxS-lacZ fusion in a luxS + merodiploid strain and evaluated its relative expression in uvrY and csrA mutants. At the entry of stationary phase, the expression of the fusion and AI-2 accumulation was positively regulated by uvrY and negatively regulated by csrA respectively. A deletion of csrA altered message stability of the luxS transcript and CsrA protein exhibited weak binding to 5' luxS regulatory region. DNA protein interaction and chromatin immunoprecipitation analysis confirmed direct interaction of UvrY with the luxS promoter. Additionally, reduced expression of the fusion in hfq deletion mutant suggested involvement of small RNA interactions in luxS regulation. In contrast, the expression of lsrA operon involved in AI-2 uptake, is negatively regulated by uvrY and positively by csrA in a cell-density dependent manner. The dual role of csrA in AI-2 synthesis and uptake suggested a regulatory crosstalk of cell signaling with carbon regulation in Escherichia coli. We found that the cAMP-CRP mediated catabolite repression of luxS expression was uvrY dependent. This study suggests that luxS expression is complex and regulated at the level of transcription and translation. The multifactorial regulation supports the notion that cell-cell communication requires interaction and integration of multiple metabolic signals.

  20. HIV integration sites and implications for maintenance of the reservoir.

    Science.gov (United States)

    Symons, Jori; Cameron, Paul U; Lewin, Sharon R

    2018-03-01

    To provide an overview of recent research of how HIV integration relates to productive and latent infection and implications for cure strategies. How and where HIV integrates provides new insights into how HIV persists on antiretroviral therapy (ART). Clonal expansion of infected cells with the same integration site demonstrates that T-cell proliferation is an important factor in HIV persistence, however, the driver of proliferation remains unclear. Clones with identical integration sites harbouring defective provirus can accumulate in HIV-infected individuals on ART and defective proviruses can express RNA and produce protein. HIV integration sites differ in clonally expanded and nonexpanded cells and in latently and productively infected cells and this influences basal and inducible transcription. There is a growing number of cellular proteins that can alter the pattern of integration to favour latency. Understanding these pathways may identify new interventions to eliminate latently infected cells. Using advances in analysing HIV integration sites, T-cell proliferation of latently infected cells is thought to play a major role in HIV persistence. Clonal expansion has been demonstrated with both defective and intact viruses. Production of viral RNA and protein from defective viruses may play a role in driving chronic immune activation. The site of integration may determine the likelihood of proliferation and the degree of basal and induced transcription. Finally, host factors and gene expression at the time of infection may determine the integration site. Together these new insights may lead to novel approaches to elimination of latently infected cells.

  1. Determinants of cell-to-cell variability in protein kinase signaling.

    Science.gov (United States)

    Jeschke, Matthias; Baumgärtner, Stephan; Legewie, Stefan

    2013-01-01

    Cells reliably sense environmental changes despite internal and external fluctuations, but the mechanisms underlying robustness remain unclear. We analyzed how fluctuations in signaling protein concentrations give rise to cell-to-cell variability in protein kinase signaling using analytical theory and numerical simulations. We characterized the dose-response behavior of signaling cascades by calculating the stimulus level at which a pathway responds ('pathway sensitivity') and the maximal activation level upon strong stimulation. Minimal kinase cascades with gradual dose-response behavior show strong variability, because the pathway sensitivity and the maximal activation level cannot be simultaneously invariant. Negative feedback regulation resolves this trade-off and coordinately reduces fluctuations in the pathway sensitivity and maximal activation. Feedbacks acting at different levels in the cascade control different aspects of the dose-response curve, thereby synergistically reducing the variability. We also investigated more complex, ultrasensitive signaling cascades capable of switch-like decision making, and found that these can be inherently robust to protein concentration fluctuations. We describe how the cell-to-cell variability of ultrasensitive signaling systems can be actively regulated, e.g., by altering the expression of phosphatase(s) or by feedback/feedforward loops. Our calculations reveal that slow transcriptional negative feedback loops allow for variability suppression while maintaining switch-like decision making. Taken together, we describe design principles of signaling cascades that promote robustness. Our results may explain why certain signaling cascades like the yeast pheromone pathway show switch-like decision making with little cell-to-cell variability.

  2. Histochemical approaches to assess cell-to-cell transmission of misfolded proteins in neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    G. Natale

    2013-03-01

    Full Text Available Formation, aggregation and transmission of abnormal proteins are common features in neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. The mechanisms underlying protein alterations in neurodegenerative diseases remain controversial. Novel findings highlighted altered protein clearing systems as common biochemical pathways which generate protein misfolding, which in turn causes protein aggregation and protein spreading. In fact, proteinaceous aggregates are prone to cell-to-cell propagation. This is reminiscent of what happens in prion disorders, where the prion protein misfolds thus forming aggregates which spread to neighbouring cells. For this reason, the term prionoids is currently used to emphasize how several misfolded proteins are transmitted in neurodegenerative diseases following this prion-like pattern. Histochemical techniques including the use of specific antibodies covering both light and electron microscopy offer a powerful tool to describe these phenomena and investigate specific molecular steps. These include: prion like protein alterations; glycation of prion-like altered proteins to form advanced glycation end-products (AGEs; mechanisms of extracellular secretion; interaction of AGEs with specific receptors placed on neighbouring cells (RAGEs. The present manuscript comments on these phenomena aimed to provide a consistent scenario of the available histochemical approaches to dissect each specific step.

  3. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  4. Reprogramming cells with synthetic proteins.

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  5. Reprogramming cells with synthetic proteins

    Science.gov (United States)

    Yang, Xiaoxiao; Malik, Vikas; Jauch, Ralf

    2015-01-01

    Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs) has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to “read” genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivo counterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies. PMID:25652623

  6. Unconventional Protein Secretion in Animal Cells.

    Science.gov (United States)

    Ng, Fanny; Tang, Bor Luen

    2016-01-01

    All eukaryotic cells secrete a range of proteins in a constitutive or regulated manner through the conventional or canonical exocytic/secretory pathway characterized by vesicular traffic from the endoplasmic reticulum, through the Golgi apparatus, and towards the plasma membrane. However, a number of proteins are secreted in an unconventional manner, which are insensitive to inhibitors of conventional exocytosis and use a route that bypasses the Golgi apparatus. These include cytosolic proteins such as fibroblast growth factor 2 (FGF2) and interleukin-1β (IL-1β), and membrane proteins that are known to also traverse to the plasma membrane by a conventional process of exocytosis, such as α integrin and the cystic fibrosis transmembrane conductor (CFTR). Mechanisms underlying unconventional protein secretion (UPS) are actively being analyzed and deciphered, and these range from an unusual form of plasma membrane translocation to vesicular processes involving the generation of exosomes and other extracellular microvesicles. In this chapter, we provide an overview on what is currently known about UPS in animal cells.

  7. Usher protein functions in hair cells and photoreceptors

    OpenAIRE

    Cosgrove, Dominic; Zallocchi, Marisa

    2013-01-01

    The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber,...

  8. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    International Nuclear Information System (INIS)

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

    2006-01-01

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia

  9. Oxidation and adduct formation of xenobiotics in a microfluidic electrochemical cell with boron doped diamond electrodes and an integrated passive gradient rotation mixer

    NARCIS (Netherlands)

    van den Brink, Floris Teunis Gerardus; Wigger, Tina; Ma, Liwei; Odijk, Mathieu; Olthuis, Wouter; Karst, U.; van den Berg, Albert

    2016-01-01

    Reactive xenobiotic metabolites and their adduct formation with biomolecules such as proteins are important to study as they can be detrimental to human health. Here, we present a microfluidic electrochemical cell with integrated micromixer to study phase I and phase II metabolism as well as protein

  10. Digestion of isolated legume cells in a stomach-duodenum model: three mechanisms limit starch and protein hydrolysis.

    Science.gov (United States)

    Bhattarai, Rewati R; Dhital, Sushil; Wu, Peng; Chen, Xiao Dong; Gidley, Michael J

    2017-07-19

    Retention of intact plant cells to the end of the small intestine leads to transport of entrapped macronutrients such as starch and protein for colonic microbial fermentation, and is a promising mechanism to increase the content of resistant starch in diets. However, the effect of gastro-intestinal bio-mechanical processing on the intactness of plant cells and the subsequent resistance to enzymatic digestion of intracellular starch and protein are not well understood. In this study, intact cells isolated from legume cotyledons are digested in a laboratory model which mimics the mechanical and biochemical conditions of the rat stomach and duodenum. The resulting digesta are characterised in terms of cell (wall) integrity as well as intracellular starch and protein hydrolysis. The cells remained essentially intact in the model with negligible (ca. 2-3%) starch or protein digestion; however when the cells were mechanically broken and digested in the model, the hydrolysis was increased to 45-50% suggesting that intact cellular structures could survive the mixing regimes in the model stomach and duodenum sufficiently to prevent digestive enzyme access. Apart from intact cell walls providing effective barrier properties, they also limit digestibility by restricting starch gelatinisation during cooking, and significant non-specific binding of α-amylase is observed to both intact and broken cell wall components, providing a third mechanism hindering starch hydrolysis. The study suggests that the preservation of intactness of plant cells, such as from legumes, could be a viable approach to achieve the targeted delivery of resistant starch to the colon.

  11. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  12. Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

    Science.gov (United States)

    Wollman, Adam J M; Leake, Mark C

    2015-01-01

    We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of

  13. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

  14. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  15. 3D Protein Dynamics in the Cell Nucleus.

    Science.gov (United States)

    Singh, Anand P; Galland, Rémi; Finch-Edmondson, Megan L; Grenci, Gianluca; Sibarita, Jean-Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E

    2017-01-10

    The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Enzymes and membrane proteins of ADSOL-preserved red blood cells

    Directory of Open Access Journals (Sweden)

    Maria Sueli Soares Leonart

    2000-03-01

    Full Text Available CONTEXT: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods. OBJECTIVE: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability. DESIGN: Biochemical evaluation designed to study red cell preservation. SETTING: São Paulo University erythrocyte metabolism referral center. SAMPLE: Six normal blood donors from the University Hospital of the Universidade Federal do Paraná, Curitiba, Brazil. MAIN MEASUREMENTS: Weekly assay of erythrocyte adenosine-5´-triphosphate (ATP, 2,3-diphosphoglycerate (2,3DPG, hexokinase (HX, phosphofructokinase (PFK, pyruvate kinase (PK, glucose-6-phosphate dehydrogenase (G-6-PD, 6-phosphogluconic dehydrogenase (6-PGD, glyceraldehyde-3-phosphate dehydrogenase (GAPD, glutathione reduc-tase (GR, glutathione peroxidase (GSHPx, plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage. RESULTS: During ADSOL preservation, erythrocyte ATP concentration decreased 60% after 5 weeks, and 90% after 10 weeks; the pH fell from 6.8 to 6.4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90% after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30% for Hx, GAPD, GR, G-6-PD and 6-PGD, 35% for PFK and GSHPx, and 45% for PK were observed. Thereafter, a uniform 10% decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE during the 14 weeks. CONCLUSION: Although the blood viability was shown to be poor

  17. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    International Nuclear Information System (INIS)

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-01-01

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  18. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Calmon, Marilia Freitas [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil); Sichero, Laura [Molecular Biology Laboratory, Centre for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo (Brazil); Boccardo, Enrique [Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo., São Paulo (Brazil); Villa, Luisa Lina [Department of Radiology and Oncology, Faculdade de Medicina, Universidade de São Paulo, São Paulo (Brazil); Rahal, Paula, E-mail: rahalp@yahoo.com.br [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil)

    2016-09-15

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  19. Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

    International Nuclear Information System (INIS)

    Brooks, J.C.; Brooks, M.

    1985-01-01

    Permeabilized cells treated with the adenosine triphosphate analog, ( 35 S)adenosine-5'-0-3(3-thiotriphosphate) ((γ- 35 S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35 S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35 S from the cells over a 20 min period. Treatment of permeabilized cells with ATPγS inhibited secretion and 35 S incorporation into the cells. Pretreatment with ATPγS resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35 S from (γ- 35 S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables

  20. The anti-cell death FNK protein protects cells from death induced by freezing and thawing

    International Nuclear Information System (INIS)

    Sudo, Kentaro; Asoh, Sadamitsu; Ohsawa, Ikuroh; Ozaki, Daiya; Yamagata, Kumi; Ito, Hiromoto; Ohta, Shigeo

    2005-01-01

    The FNK protein, constructed from anti-apoptotic Bcl-x L with enhanced activity, was fused with the protein transduction domain (PTD) of the HIV/Tat protein to mediate the delivery of FNK into cells. The fusion protein PTD-FNK was introduced into chondrocytes in isolated articular cartilage-bone sections, cultured neurons, and isolated bone marrow mononuclear cells to evaluate its ability to prevent cell death induced by freezing and thawing. PTD-FNK protected the cells from freeze-thaw damage in a concentration-dependent manner. Addition of PTD-FNK with conventional cryoprotectants (dimethyl sulfoxide and hydroxyethyl starch) increased surviving cell numbers around 2-fold compared with controls treated only with the cryoprotectants. Notably, PTD-FNK allowed CD34 + cells among bone marrow mononuclear cells to survive more efficiently (12-fold more than the control cells) from two successive freeze-thaw cycles. Thus, PTD-FNK prevented cell death induced by freezing and thawing, suggesting that it provides for the successful cryopreservation of biological materials

  1. Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

    DEFF Research Database (Denmark)

    Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori

    2017-01-01

    , an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood...... a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice....

  2. Host cell proteins in biotechnology-derived products: A risk assessment framework.

    Science.gov (United States)

    de Zafra, Christina L Zuch; Quarmby, Valerie; Francissen, Kathleen; Vanderlaan, Martin; Zhu-Shimoni, Judith

    2015-11-01

    To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins. © 2015 Wiley Periodicals, Inc.

  3. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

    International Nuclear Information System (INIS)

    Sakai, Tomomi; Tochio, Hidehito; Tenno, Takeshi; Ito, Yutaka; Kokubo, Tetsuro; Hiroaki, Hidekazu; Shirakawa, Masahiro

    2006-01-01

    In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. 15 N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional 1 H- 15 N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions

  4. Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

    International Nuclear Information System (INIS)

    Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

    2007-01-01

    Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and β-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 μM sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and β-catenin staining at cell-cell junctions that were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of β-catenin and activation of protein kinase Cα (PKCα) were observed. Inhibition of PKCα restored VE-cadherin and β-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of β-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKCα, which leads to increased PY of β-catenin downstream of PKCα activation. Phosphorylation of β-catenin plausibly severs the association of VE-cadherin and β-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis

  5. Differential biotin labelling of the cell envelope proteins in lipopolysaccharidic diderm bacteria: Exploring the proteosurfaceome of Escherichia coli using sulfo-NHS-SS-biotin and sulfo-NHS-PEG4-bismannose-SS-biotin.

    Science.gov (United States)

    Monteiro, Ricardo; Chafsey, Ingrid; Leroy, Sabine; Chambon, Christophe; Hébraud, Michel; Livrelli, Valérie; Pizza, Mariagrazia; Pezzicoli, Alfredo; Desvaux, Mickaël

    2018-06-15

    Surface proteins are the major factor for the interaction between bacteria and its environment, playing an important role in infection, colonisation, virulence and adaptation. However, the study of surface proteins has proven difficult mainly due to their hydrophobicity and/or relatively low abundance compared with cytoplasmic proteins. To overcome these issues new proteomic strategies have been developed, such as cell-surface protein labelling using biotinylation reagents. Sulfo-NHS-SS-biotin is the most commonly used reagent to investigate the proteins expressed at the cell surface of various organisms but its use in lipopolysaccharidic diderm bacteria (archetypical Gram-negative bacteria) remains limited to a handful of species. While generally pass over in silence, some periplasmic proteins, but also some inner membrane lipoproteins, integral membrane proteins and cytoplasmic proteins (cytoproteins) are systematically identified following this approach. To limit cell lysis and diffusion of the sulfo-NHS-SS-biotin through the outer membrane, biotin labelling was tested over short incubation times and proved to be as efficient for 1 min at room temperature. To further limit labelling of protein located below the outer membrane, the use of high-molecular weight sulfo-NHS-PEG4-bismannose-SS-biotin appeared to recover differentially cell-envelope proteins compared to low-molecular weight sulfo-NHS-SS-biotin. Actually, the sulfo-NHS-SS-biotin recovers at a higher extent the proteins completely or partly exposed in the periplasm than sulfo-NHS-PEG4-bismannose-SS-biotin, namely periplasmic and integral membrane proteins as well as inner membrane and outer membrane lipoproteins. These results highlight that protein labelling using biotinylation reagents of different sizes provides a sophisticated and accurate way to differentially explore the cell envelope proteome of lipopolysaccharidic diderm bacteria. While generally pass over in silence, some periplasmic proteins

  6. Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.

    Science.gov (United States)

    Xia, Yan; Fischer, Axel W; Teixeira, Pedro; Weiner, Brian; Meiler, Jens

    2018-04-03

    While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Determinants of cell-to-cell variability in protein kinase signaling.

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    Matthias Jeschke

    Full Text Available Cells reliably sense environmental changes despite internal and external fluctuations, but the mechanisms underlying robustness remain unclear. We analyzed how fluctuations in signaling protein concentrations give rise to cell-to-cell variability in protein kinase signaling using analytical theory and numerical simulations. We characterized the dose-response behavior of signaling cascades by calculating the stimulus level at which a pathway responds ('pathway sensitivity' and the maximal activation level upon strong stimulation. Minimal kinase cascades with gradual dose-response behavior show strong variability, because the pathway sensitivity and the maximal activation level cannot be simultaneously invariant. Negative feedback regulation resolves this trade-off and coordinately reduces fluctuations in the pathway sensitivity and maximal activation. Feedbacks acting at different levels in the cascade control different aspects of the dose-response curve, thereby synergistically reducing the variability. We also investigated more complex, ultrasensitive signaling cascades capable of switch-like decision making, and found that these can be inherently robust to protein concentration fluctuations. We describe how the cell-to-cell variability of ultrasensitive signaling systems can be actively regulated, e.g., by altering the expression of phosphatase(s or by feedback/feedforward loops. Our calculations reveal that slow transcriptional negative feedback loops allow for variability suppression while maintaining switch-like decision making. Taken together, we describe design principles of signaling cascades that promote robustness. Our results may explain why certain signaling cascades like the yeast pheromone pathway show switch-like decision making with little cell-to-cell variability.

  8. Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

    2003-01-01

    Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

  9. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

    Directory of Open Access Journals (Sweden)

    Feng Gao

    2016-08-01

    Full Text Available Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag, by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

  10. Semantic integration to identify overlapping functional modules in protein interaction networks

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    Ramanathan Murali

    2007-07-01

    Full Text Available Abstract Background The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms. Results We have developed novel metrics, called semantic similarity and semantic interactivity, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. We presented a flow-based modularization algorithm to efficiently identify overlapping modules in the weighted interaction networks. The experimental results show that the semantic similarity and semantic interactivity of interacting pairs were positively correlated with functional co-occurrence. The effectiveness of the algorithm for identifying modules was evaluated using functional categories from the MIPS database. We demonstrated that our algorithm had higher accuracy compared to other competing approaches. Conclusion The integration of protein interaction networks with GO annotation data and the capability of detecting overlapping modules substantially improve the accuracy of module identification.

  11. Cellular Reprogramming Using Protein and Cell-Penetrating Peptides

    Directory of Open Access Journals (Sweden)

    Bong Jong Seo

    2017-03-01

    Full Text Available Recently, stem cells have been suggested as invaluable tools for cell therapy because of their self-renewal and multilineage differentiation potential. Thus, scientists have developed a variety of methods to generate pluripotent stem cells, from nuclear transfer technology to direct reprogramming using defined factors, or induced pluripotent stem cells (iPSCs. Considering the ethical issues and efficiency, iPSCs are thought to be one of the most promising stem cells for cell therapy. Induced pluripotent stem cells can be generated by transduction with a virus, plasmid, RNA, or protein. Herein, we provide an overview of the current technology for iPSC generation and describe protein-based transduction technology in detail.

  12. In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

    Science.gov (United States)

    Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

    2015-08-04

    Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

  13. Thematic minireview series: cell biology of G protein signaling.

    Science.gov (United States)

    Dohlman, Henrik G

    2015-03-13

    This thematic series is on the topic of cell signaling from a cell biology perspective, with a particular focus on G proteins. G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors) are typically found at the cell surface. Upon agonist binding, these receptors will activate a GTP-binding G protein at the cytoplasmic face of the plasma membrane. Additionally, there is growing evidence that G proteins can also be activated by non-receptor binding partners, and they can signal from non-plasma membrane compartments. The production of second messengers at multiple, spatially distinct locations represents a type of signal encoding that has been largely neglected. The first minireview in the series describes biosensors that are being used to monitor G protein signaling events in live cells. The second describes the implementation of antibody-based biosensors to dissect endosome signaling by G proteins and their receptors. The third describes the function of a non-receptor, cytoplasmic activator of G protein signaling, called GIV (Girdin). Collectively, the advances described in these articles provide a deeper understanding and emerging opportunities for new pharmacology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Integral membrane protein structure determination using pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

    2015-04-15

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

  15. Prolactin-inducible proteins in human breast cancer cells

    International Nuclear Information System (INIS)

    Shiu, R.P.; Iwasiow, B.M.

    1985-01-01

    The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [ 35 S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [ 3 H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11

  16. Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

    Science.gov (United States)

    Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

    2017-06-01

    RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

  17. Growing functional modules from a seed protein via integration of protein interaction and gene expression data

    Directory of Open Access Journals (Sweden)

    Dimitrakopoulou Konstantina

    2007-10-01

    Full Text Available Abstract Background Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. Results In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. Conclusion The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

  18. General theory for integrated analysis of growth, gene, and protein expression in biofilms.

    Science.gov (United States)

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.

  19. Global Conservation of Protein Status between Cell Lines and Xenografts

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    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  20. B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells

    International Nuclear Information System (INIS)

    Wu, Qiang; Kong, Xiang-jun; Xu, Xiao-chun; Lobie, Peter E; Zhu, Tao; Wu, Zheng-sheng; Liu, Xue; Yan, Hong; He, Yin-huan; Ye, Shan; Cheng, Xing-wang; Zhu, Gui-lu; Wu, Wen-yong; Wang, Xiao-nan

    2014-01-01

    B-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients. Expression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells. BCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins. The current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer

  1. ASIC proteins regulate smooth muscle cell migration.

    Science.gov (United States)

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

  2. Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

    Science.gov (United States)

    Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

    2016-01-01

    The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an

  3. Three mitogen-activated protein kinases required for cell wall integrity contribute greatly to biocontrol potential of a fungal entomopathogen.

    Directory of Open Access Journals (Sweden)

    Ying Chen

    Full Text Available Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP kinases constituting cell wall integrity (CWI pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

  4. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Science.gov (United States)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  5. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

    International Nuclear Information System (INIS)

    Apostolou, Andria; Shen Yuxian; Liang Yan; Luo Jun; Fang Shengyun

    2008-01-01

    The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR

  6. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    Science.gov (United States)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  7. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  8. Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

    2017-10-07

    The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

    Science.gov (United States)

    Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

    2013-07-19

    Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

  10. Induction of the unfolded protein response by constitutive G-protein signaling in rod photoreceptor cells.

    Science.gov (United States)

    Wang, Tian; Chen, Jeannie

    2014-10-17

    Phototransduction is a G-protein signal transduction cascade that converts photon absorption to a change in current at the plasma membrane. Certain genetic mutations affecting the proteins in the phototransduction cascade cause blinding disorders in humans. Some of these mutations serve as a genetic source of "equivalent light" that activates the cascade, whereas other mutations lead to amplification of the light response. How constitutive phototransduction causes photoreceptor cell death is poorly understood. We showed that persistent G-protein signaling, which occurs in rod arrestin and rhodopsin kinase knock-out mice, caused a rapid and specific induction of the PERK pathway of the unfolded protein response. These changes were not observed in the cGMP-gated channel knock-out rods, an equivalent light condition that mimics light-stimulated channel closure. Thus transducin signaling, but not channel closure, triggers rapid cell death in light damage caused by constitutive phototransduction. Additionally, we show that in the albino light damage model cell death was not associated with increase in global protein ubiquitination or unfolded protein response induction. Taken together, these observations provide novel mechanistic insights into the cell death pathway caused by constitutive phototransduction and identify the unfolded protein response as a potential target for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Integrated modulation of phorbol ester-induced Raf activation in EL4 lymphoma cells.

    Science.gov (United States)

    Han, Shujie; Meier, Kathryn E

    2009-05-01

    The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that "PMA-sensitive" cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In "PMA-resistant" and "intermediate" EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation.

  12. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  13. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Melia, Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden (Netherlands); Marrison, Jo [Department of Biology, The University of York, Heslington, York (United Kingdom); Carzaniga, Raffaella [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Domart, Marie-Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); O' Toole, Peter [Department of Biology, The University of York, Heslington, York (United Kingdom); Larijani, Banafshe [Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa (Spain); IKERBASQUE, Basque Foundation for Science, Bilbao (Spain); Collinson, Lucy M. [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom)

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. - Highlights: • GFP and mCherry fluorescence are preserved in heavy-metal stained mammalian cells embedded in resin • Fluorophores are stable and intensity is sufficient for detection in ultrathin sections • Overlay of separate LM and EM images from the same ultrathin section improves CLEM protein localisation precision • GFP is stable and active in the vacuum of an integrated light and scanning EM • Integrated light and electron microscopy shows new subcellular locations of the lipid diacylglycerol.

  14. The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

    Science.gov (United States)

    Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

    2007-10-01

    MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

  15. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Fogeron, Marie-Laure [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Jirasko, Vlastimil; Penzel, Susanne [ETH Zurich, Physical Chemistry (Switzerland); Paul, David [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Montserret, Roland; Danis, Clément; Lacabanne, Denis; Badillo, Aurélie [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Gouttenoire, Jérôme; Moradpour, Darius [University of Lausanne, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois (Switzerland); Bartenschlager, Ralf [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Penin, François [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); and others

    2016-06-15

    We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [{sup 2}H,{sup 13}C,{sup 15}N]-labeled protein are shown to yield narrow {sup 13}C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

  16. Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

    Science.gov (United States)

    Spens, Erika; Häggström, Lena

    2009-05-20

    NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

  17. Cell wall proteins in seedling cotyledons of Prosopis chilensis.

    Science.gov (United States)

    Rodríguez, J G; Cardemil, L

    1994-01-01

    Four cell wall proteins of cotyledons of Prosopis chilensis seedlings were characterized by PAGE and Western analyses using a polyclonal antibody, generated against soybean seed coat extensin. These proteins had M(r)s of 180,000, 126,000, 107,000 and 63,000, as determined by SDS-PAGE. The proteins exhibited a fluorescent positive reaction with dansylhydrazine suggesting that they are glycoproteins; they did not show peroxidase activity. The cell wall proteins were also characterized by their amino acid composition and by their amino-terminal sequence. These analyses revealed that there are two groups of related cell wall proteins in the cotyledons. The first group comprises the proteins of M(r)s 180,000, 126,000, 107,000 which are rich in glutamic acid/glutamine and aspartic acid/asparagine and they have almost identical NH2-terminal sequences. The second group comprises the M(r) 63,000 protein which is rich in proline, glycine, valine and tyrosine, with an NH2-terminal sequence which was very similar to that of soybean proline-rich proteins.

  18. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    International Nuclear Information System (INIS)

    Beebe, D.P.; Wood, L.L.; Moos, M.

    1990-01-01

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between 125 I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase

  19. In vitro protein expression: an emerging alternative to cell-based approaches.

    Science.gov (United States)

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Protein synthesis and sublethal damage repair in synchronized CHO cells

    International Nuclear Information System (INIS)

    Yezzi, M.J.; Tobias, C.A.; Blakely, E.A.

    1984-01-01

    The authors have previously reported that the split dose survival response to x-rays of asynchronous CHO-TSH1 cells is reduced if the cells are held at 40 0 C,a temperature that inhibits protein synthesis, for 2 hours before the first dose and during a 2-hour interval between doses. In conjunction with the survival experiments on asynchronous cells, the authors also examined the DNA rejoining ability in split dose studies with and without inhibition of protein synthesis. The results of these experiments suggest that inhibition of protein synthesis affects a pool of proteins that are necessary for the correct expression of the DNA, although they do not appear to be involved in rejoining DNA breaks. They have extended this work to the study of cells synchronized in G1 phase (2 hour post-mitosis) and S phase (10 hour post-mitosis). Autoradiographic analyses, using 3H-TdR pulse labeling, demonstrated that a delay in the progression of each synchronized cell population occurs after inhibition of protein synthesis. Data are reported on the effects of inhibition of protein synthesis on the ability of G1 and S phase cells to repair sublethal damage

  1. Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

    Science.gov (United States)

    Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

    2017-07-07

    Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Integrative approaches to the prediction of protein functions based on the feature selection

    Directory of Open Access Journals (Sweden)

    Lee Hyunju

    2009-12-01

    Full Text Available Abstract Background Protein function prediction has been one of the most important issues in functional genomics. With the current availability of various genomic data sets, many researchers have attempted to develop integration models that combine all available genomic data for protein function prediction. These efforts have resulted in the improvement of prediction quality and the extension of prediction coverage. However, it has also been observed that integrating more data sources does not always increase the prediction quality. Therefore, selecting data sources that highly contribute to the protein function prediction has become an important issue. Results We present systematic feature selection methods that assess the contribution of genome-wide data sets to predict protein functions and then investigate the relationship between genomic data sources and protein functions. In this study, we use ten different genomic data sources in Mus musculus, including: protein-domains, protein-protein interactions, gene expressions, phenotype ontology, phylogenetic profiles and disease data sources to predict protein functions that are labelled with Gene Ontology (GO terms. We then apply two approaches to feature selection: exhaustive search feature selection using a kernel based logistic regression (KLR, and a kernel based L1-norm regularized logistic regression (KL1LR. In the first approach, we exhaustively measure the contribution of each data set for each function based on its prediction quality. In the second approach, we use the estimated coefficients of features as measures of contribution of data sources. Our results show that the proposed methods improve the prediction quality compared to the full integration of all data sources and other filter-based feature selection methods. We also show that contributing data sources can differ depending on the protein function. Furthermore, we observe that highly contributing data sets can be similar among

  3. Finding undetected protein associations in cell signaling by belief propagation.

    Science.gov (United States)

    Bailly-Bechet, M; Borgs, C; Braunstein, A; Chayes, J; Dagkessamanskaia, A; François, J-M; Zecchina, R

    2011-01-11

    External information propagates in the cell mainly through signaling cascades and transcriptional activation, allowing it to react to a wide spectrum of environmental changes. High-throughput experiments identify numerous molecular components of such cascades that may, however, interact through unknown partners. Some of them may be detected using data coming from the integration of a protein-protein interaction network and mRNA expression profiles. This inference problem can be mapped onto the problem of finding appropriate optimal connected subgraphs of a network defined by these datasets. The optimization procedure turns out to be computationally intractable in general. Here we present a new distributed algorithm for this task, inspired from statistical physics, and apply this scheme to alpha factor and drug perturbations data in yeast. We identify the role of the COS8 protein, a member of a gene family of previously unknown function, and validate the results by genetic experiments. The algorithm we present is specially suited for very large datasets, can run in parallel, and can be adapted to other problems in systems biology. On renowned benchmarks it outperforms other algorithms in the field.

  4. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization and Pathogenicity

    Directory of Open Access Journals (Sweden)

    Wei Wei

    2016-08-01

    Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homologue in C. lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum.

  5. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

    Science.gov (United States)

    Levin, David E.

    2011-01-01

    The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

  6. The base excision repair pathway is required for efficient lentivirus integration.

    Directory of Open Access Journals (Sweden)

    Kristine E Yoder

    Full Text Available An siRNA screen has identified several proteins throughout the base excision repair (BER pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.

  7. Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

    International Nuclear Information System (INIS)

    Born, E J; Hartman, S V; Holstein, S A

    2013-01-01

    Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

  8. Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

    KAUST Repository

    Haidar, Malak

    2017-09-08

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

  9. Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Hyun Kim

    2012-05-01

    Full Text Available Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

  10. Predicting co-complexed protein pairs using genomic and proteomic data integration

    Directory of Open Access Journals (Sweden)

    King Oliver D

    2004-04-01

    Full Text Available Abstract Background Identifying all protein-protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H and affinity purification coupled with mass spectrometry (APMS have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship. Results Using a supervised machine learning approach – probabilistic decision tree, we integrated high-throughput protein interaction datasets and other gene- and protein-pair characteristics to predict co-complexed pairs (CCP of proteins. Our predictions proved more sensitive and specific than predictions based on Y2H or APMS methods alone or in combination. Among the top predictions not annotated as CCPs in our reference set (obtained from the MIPS complex catalogue, a significant fraction was found to physically interact according to a separate database (YPD, Yeast Proteome Database, and the remaining predictions may potentially represent unknown CCPs. Conclusions We demonstrated that the probabilistic decision tree approach can be successfully used to predict co-complexed protein (CCP pairs from other characteristics. Our top-scoring CCP predictions provide testable hypotheses for experimental validation.

  11. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. The outer membrane protein assembly machinery of Neisseria meningitidis

    NARCIS (Netherlands)

    Volokhina, E.B.|info:eu-repo/dai/nl/304837202

    2009-01-01

    Gram-negative bacteria are characterized by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), which are separated by the peptidoglycan-containing periplasm. While the integral IM proteins are alpha-helical, all but one known integral OM proteins (OMPs) are

  13. Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

    International Nuclear Information System (INIS)

    Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

    1984-01-01

    Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)

  14. What befalls the proteins and water in a living cell when the cell dies?

    Science.gov (United States)

    Ling, Gilbert N; Fu, Ya-zhen

    2005-01-01

    The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.

  15. Diversity of T cell epitopes in Plasmodium falciparum circumsporozoite protein likely due to protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Nagesh R Aragam

    Full Text Available Circumsporozoite protein (CS is a leading vaccine antigen for falciparum malaria, but is highly polymorphic in natural parasite populations. The factors driving this diversity are unclear, but non-random assortment of the T cell epitopes TH2 and TH3 has been observed in a Kenyan parasite population. The recent publication of the crystal structure of the variable C terminal region of the protein allows the assessment of the impact of diversity on protein structure and T cell epitope assortment. Using data from the Gambia (55 isolates and Malawi (235 isolates, we evaluated the patterns of diversity within and between epitopes in these two distantly-separated populations. Only non-synonymous mutations were observed with the vast majority in both populations at similar frequencies suggesting strong selection on this region. A non-random pattern of T cell epitope assortment was seen in Malawi and in the Gambia, but structural analysis indicates no intramolecular spatial interactions. Using the information from these parasite populations, structural analysis reveals that polymorphic amino acids within TH2 and TH3 colocalize to one side of the protein, surround, but do not involve, the hydrophobic pocket in CS, and predominately involve charge switches. In addition, free energy analysis suggests residues forming and behind the novel pocket within CS are tightly constrained and well conserved in all alleles. In addition, free energy analysis shows polymorphic residues tend to be populated by energetically unfavorable amino acids. In combination, these findings suggest the diversity of T cell epitopes in CS may be primarily an evolutionary response to intermolecular interactions at the surface of the protein potentially counteracting antibody-mediated immune recognition or evolving host receptor diversity.

  16. Membrane Proteins : The Key Players of a Cancer Cell

    NARCIS (Netherlands)

    Kampen, Kim R.

    Membrane proteins are involved in the prognosis of the most common forms of cancer. Membrane proteins are the hallmark of a cancer cell. The overexpressed membrane receptors are becoming increasingly important in cancer cell therapy. Current renewing therapy approaches based on receptor

  17. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

    Directory of Open Access Journals (Sweden)

    Lena Thoring

    Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

  18. PANDORA: keyword-based analysis of protein sets by integration of annotation sources.

    Science.gov (United States)

    Kaplan, Noam; Vaaknin, Avishay; Linial, Michal

    2003-10-01

    Recent advances in high-throughput methods and the application of computational tools for automatic classification of proteins have made it possible to carry out large-scale proteomic analyses. Biological analysis and interpretation of sets of proteins is a time-consuming undertaking carried out manually by experts. We have developed PANDORA (Protein ANnotation Diagram ORiented Analysis), a web-based tool that provides an automatic representation of the biological knowledge associated with any set of proteins. PANDORA uses a unique approach of keyword-based graphical analysis that focuses on detecting subsets of proteins that share unique biological properties and the intersections of such sets. PANDORA currently supports SwissProt keywords, NCBI Taxonomy, InterPro entries and the hierarchical classification terms from ENZYME, SCOP and GO databases. The integrated study of several annotation sources simultaneously allows a representation of biological relations of structure, function, cellular location, taxonomy, domains and motifs. PANDORA is also integrated into the ProtoNet system, thus allowing testing thousands of automatically generated clusters. We illustrate how PANDORA enhances the biological understanding of large, non-uniform sets of proteins originating from experimental and computational sources, without the need for prior biological knowledge on individual proteins.

  19. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  20. Inhibition of host cell protein synthesis by UV-inactivated poliovirus

    International Nuclear Information System (INIS)

    Helentjaris, T.; Ehrenfeld, E.

    1977-01-01

    The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm 2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell

  1. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    International Nuclear Information System (INIS)

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

    2014-01-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

  2. Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

    Directory of Open Access Journals (Sweden)

    Lishi Xie

    Full Text Available Protein Kinase C (PKC plays a significant role in thrombin-induced loss of endothelial cell (EC barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin, dominant negative PKCδ construct and PKCδ silencing (siRNA. In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.

  3. Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

    Science.gov (United States)

    2014-01-01

    Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

  4. Dendritic cell nuclear protein-1, a novel depression-related protein, upregulates corticotropin-releasing hormone expression

    NARCIS (Netherlands)

    Zhou, Tian; Wang, Shanshan; Ren, Haigang; Qi, Xin-Rui; Luchetti, Sabina; Kamphuis, Willem; Zhou, Jiang-Ning; Wang, Guanghui; Swaab, Dick F.

    2010-01-01

    The recently discovered dendritic cell nuclear protein-1 is the product of a novel candidate gene for major depression. The A allele encodes full-length dendritic cell nuclear protein-1, while the T allele encodes a premature termination of translation at codon number 117 on chromosome 5. In the

  5. Unveiling network-based functional features through integration of gene expression into protein networks.

    Science.gov (United States)

    Jalili, Mahdi; Gebhardt, Tom; Wolkenhauer, Olaf; Salehzadeh-Yazdi, Ali

    2018-06-01

    Decoding health and disease phenotypes is one of the fundamental objectives in biomedicine. Whereas high-throughput omics approaches are available, it is evident that any single omics approach might not be adequate to capture the complexity of phenotypes. Therefore, integrated multi-omics approaches have been used to unravel genotype-phenotype relationships such as global regulatory mechanisms and complex metabolic networks in different eukaryotic organisms. Some of the progress and challenges associated with integrated omics studies have been reviewed previously in comprehensive studies. In this work, we highlight and review the progress, challenges and advantages associated with emerging approaches, integrating gene expression and protein-protein interaction networks to unravel network-based functional features. This includes identifying disease related genes, gene prioritization, clustering protein interactions, developing the modules, extract active subnetworks and static protein complexes or dynamic/temporal protein complexes. We also discuss how these approaches contribute to our understanding of the biology of complex traits and diseases. This article is part of a Special Issue entitled: Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Selective radiolabeling of cell surface proteins to a high specific activity

    International Nuclear Information System (INIS)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-01-01

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125 I-(hydroxyphenyl)propionyl groups via 125 I-sulfosuccinimidyl (hydroxyphenyl)propionate ( 125 II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[ 125 I]iodide labeling technique revealed that 125 I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125 I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane

  7. MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

    Science.gov (United States)

    Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

    2018-02-02

    Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

  8. In-cell thermodynamics and a new role for protein surfaces.

    Science.gov (United States)

    Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

    2016-02-16

    There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

  9. Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

    Science.gov (United States)

    Suk, Hyung; Knipe, David M

    2015-06-01

    The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Protein carbonylation, protein aggregation and neuronal cell death in a murine model of multiple sclerosis

    Science.gov (United States)

    Dasgupta, Anushka

    Many studies have suggested that oxidative stress plays an important role in the pathophysiology of both multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Yet, the mechanism by which oxidative stress leads to tissue damage in these disorders is unclear. Recent work from our laboratory has revealed that protein carbonylation, a major oxidative modification caused by severe and/or chronic oxidative stress conditions, is elevated in MS and EAE. Furthermore, protein carbonylation has been shown to alter protein structure leading to misfolding/aggregation. These findings prompted me to hypothesize that carbonylated proteins, formed as a consequence of oxidative stress and/or decreased proteasomal activity, promote protein aggregation to mediate neuronal apoptosis in vitro and in EAE. To test this novel hypothesis, I first characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of myelin-oligodendrocyte glycoprotein (MOG)35-55 peptide-induced EAE in C57BL/6 mice [Chapter 2]. The results show that carbonylated proteins accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. I discovered not only that there is a temporal correlation between protein carbonylation and apoptosis but also that carbonyl levels are significantly higher in apoptotic cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are also present during the course of EAE, which seems to be due to reduced autophagy. In chapter 3, I show that when gluthathione levels are reduced to those in EAE spinal cord, both neuron-like PC12 (nPC12) cells and primary neuronal cultures accumulate carbonylated proteins and undergo cell death (both by necrosis and apoptosis). Immunocytochemical and biochemical studies also revealed a temporal

  11. Nuclear transport of heat shock proteins in stressed cells

    International Nuclear Information System (INIS)

    Chughtai, Zahoor Saeed

    2001-01-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or β-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay I have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei of stationary phase cells. (author)

  12. Nuclear transport of heat shock proteins in stressed cells

    Energy Technology Data Exchange (ETDEWEB)

    Chughtai, Zahoor Saeed

    2001-07-01

    Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy , and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the heat shock protein hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of nutrient-depleted cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. Upon starvation, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or {beta}-gaIactosidase to nuclei. To determine whether nuclear accumulation of Star-{beta}-galactosidase depends on a specific nuclear carrier, I have analyzed its distribution in mutant yeast strains that carry a deletion of a single {beta}-importin gene. With this assay I have identified Nmd5p as a {beta}-importin required to concentrate Star-{beta}-galactosidase in nuclei of stationary phase cells. (author)

  13. The Tobacco mosaic virus Movement Protein Associates with but Does Not Integrate into Biological Membranes

    Science.gov (United States)

    Peiró, Ana; Martínez-Gil, Luis; Tamborero, Silvia; Pallás, Vicente

    2014-01-01

    ABSTRACT Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport

  14. Chaperone-protease networks in mitochondrial protein homeostasis.

    Science.gov (United States)

    Voos, Wolfgang

    2013-02-01

    As essential organelles, mitochondria are intimately integrated into the metabolism of a eukaryotic cell. The maintenance of the functional integrity of the mitochondrial proteome, also termed protein homeostasis, is facing many challenges both under normal and pathological conditions. First, since mitochondria are derived from bacterial ancestor cells, the proteins in this endosymbiotic organelle have a mixed origin. Only a few proteins are encoded on the mitochondrial genome, most genes for mitochondrial proteins reside in the nuclear genome of the host cell. This distribution requires a complex biogenesis of mitochondrial proteins, which are mostly synthesized in the cytosol and need to be imported into the organelle. Mitochondrial protein biogenesis usually therefore comprises complex folding and assembly processes to reach an enzymatically active state. In addition, specific protein quality control (PQC) processes avoid an accumulation of damaged or surplus polypeptides. Mitochondrial protein homeostasis is based on endogenous enzymatic components comprising a diverse set of chaperones and proteases that form an interconnected functional network. This review describes the different types of mitochondrial proteins with chaperone functions and covers the current knowledge of their roles in protein biogenesis, folding, proteolytic removal and prevention of aggregation, the principal reactions of protein homeostasis. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

    Science.gov (United States)

    Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

    2017-10-01

    Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

  16. Respiratory epithelial cell responses to cigarette smoke: the unfolded protein response.

    Science.gov (United States)

    Kelsen, Steven G

    2012-12-01

    Cigarette smoking exposes the respiratory epithelium to highly toxic, reactive oxygen nitrogen species which damage lung proteins in the endoplasmic reticulum (ER), the cell organelle in which all secreted and membrane proteins are processed. Accumulation of damaged or misfolded proteins in the ER, a condition termed ER stress, activates a complex cellular process termed the unfolded protein responses (UPR). The UPR acts to restore cellular protein homeostasis by regulating all aspects of protein metabolism including: protein translation and syntheses; protein folding; and protein degradation. However, activation of the UPR may also induce signaling pathways which induce inflammation and cell apoptosis. This review discusses the role of UPR in the respiratory epithelial cell response to cigarette smoke and the pathogenesis of lung diseases like COPD. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine.

    Science.gov (United States)

    Aldarouish, Mohanad; Wang, Huzhan; Zhou, Meng; Hu, Hong-Ming; Wang, Li-Xin

    2015-04-16

    Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine. The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis. We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub

  18. Biosynthesis of proteins and radiation effects in cells

    International Nuclear Information System (INIS)

    Kolomiets, K.D.

    1982-01-01

    Critical analysis of nowadays literature and own experimental data on importance of biosynthesis of proteins, their modification and functional activity in forming radiation effects in irradiated cells is given. A special place in the development of radiation injury of cellular structures and in reduction processes is allocated to molecular recognition. The data on the role of protein synthesis and molecular recognition in the reduction of main biological cell chromatin system are presented. The dependence of postradiation changes in the cell on structural and functional chromatin state is considered

  19. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    Directory of Open Access Journals (Sweden)

    Girolamo Antonietta

    2011-05-01

    Full Text Available Abstract Background The MP65 gene of Candida albicans (orf19.1779 encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p, a high level of expression of two stress-related genes (DDR48 and SOD5, and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

  20. Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

    Science.gov (United States)

    Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

    2013-01-01

    Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

  1. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    Science.gov (United States)

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  2. MULTIFUNCTIONAL ADHESIN PROTEINS AND THEIR DISPLAY IN MICROBIAL CELLS

    DEFF Research Database (Denmark)

    1999-01-01

    Recombinant cells expressing a multifunctional adhesin protein derived from a naturally occurring adhesin, containing a binding domain that is capable of binding to an organic receptor and a binding domain that is capable of binding to a compound to which the naturally occurring adhesin protein...... substantially does not bind. The cells or modified adhesin proteins, optionally in immobilized form, are useful for separating organic and inorganic compounds including toxic or precious metals from an environment....

  3. Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

    Science.gov (United States)

    Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2018-06-27

    The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

  4. AN INTEGRATIVE WAY OF TEACHING MOLECULAR CELL BIOLOGY AND PROTEIN CHEMISTRY USING ACTIN IMMOBILIZATION ON CHITIN FOR PURIFYING MYOSIN II.

    Directory of Open Access Journals (Sweden)

    M.G. Souza

    2007-05-01

    Full Text Available Our intent is to present our experience on teaching Molecular Cell Biology andProtein Chemistry at UNIRIO through an innovative approach that includes myosin IIextraction and purification. We took advantage of the properties of muscle contractionand propose a simple method for purifying myosin II by affinity chromatography. Thisoriginal method is based on the preparation of an affinity column containing actinmolecules covalently bound to chitin particles. We propose a three-week syllabus thatincludes lectures and bench experimental work. The syllabus favors the activelearning of protein extraction and purification, as well as, of scientific concepts suchas muscle contraction, cytoskeleton structure and its importance for the living cell. Italso promotes the learning of the biotechnological applications of chitin and theapplications of protein immobilization in different industrial fields. Furthermore, theactivities also target the development of laboratorial technical abilities, thedevelopment of problem solving skills and the ability to write up a scientific reportfollowing the model of a scientific article. It is very important to mention that thissyllabus can be used even in places where a facility such as ultra-centrifugation islacking.

  5. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  6. Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

    Science.gov (United States)

    Hirayama, Mio; Kobayashi, Daiki; Mizuguchi, Souhei; Morikawa, Takashi; Nagayama, Megumi; Midorikawa, Uichi; Wilson, Masayo M; Nambu, Akiko N; Yoshizawa, Akiyasu C; Kawano, Shin; Araki, Norie

    2013-05-01

    Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural

  7. Direct digestion of proteins in living cells into peptides for proteomic analysis.

    Science.gov (United States)

    Chen, Qi; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2015-01-01

    To analyze the proteome of an extremely low number of cells or even a single cell, we established a new method of digesting whole cells into mass-spectrometry-identifiable peptides in a single step within 2 h. Our sampling method greatly simplified the processes of cell lysis, protein extraction, protein purification, and overnight digestion, without compromising efficiency. We used our method to digest hundred-scale cells. As far as we know, there is no report of proteome analysis starting directly with as few as 100 cells. We identified an average of 109 proteins from 100 cells, and with three replicates, the number of proteins rose to 204. Good reproducibility was achieved, showing stability and reliability of the method. Gene Ontology analysis revealed that proteins in different cellular compartments were well represented.

  8. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Niels Bjerg; Strucko, Tomas; Kildegaard, Kanchana Rueksomtawin

    2014-01-01

    of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red...... fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded...... on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain...

  9. Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

    Science.gov (United States)

    Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

    2018-01-01

    Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

  10. ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

    Science.gov (United States)

    Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

    2013-03-01

    Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

  11. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    OpenAIRE

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  12. Regulation of the vertebrate cell cycle by the cdc2 protein kinase

    International Nuclear Information System (INIS)

    Draetta, G.; Brizuela, L.; Moran, B.; Beach, D.

    1988-01-01

    A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. The authors have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. They find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G 1 but displays maximal enzymatic activity during mitotic metaphase. These observations indicate that in a wide variety of vertebrate cells, the cdc2 protein kinase is involved in regulating mitosis. The authors' approach taken toward study of the cdc2 protein kinase highlights the possibilities that now exist for combining the advantages of ascomycete genetics with the cell-free systems of Xenopus and the biochemical advantages of tissue culture cells to investigate fundamental problems of the cell cycle

  13. Algal autolysate medium to label proteins for NMR in mammalian cells.

    Science.gov (United States)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  14. Algal autolysate medium to label proteins for NMR in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia [University of Florence, Magnetic Resonance Center (CERM) (Italy); Neri, Sara [Giotto Biotech S.R.L. (Italy); Fragai, Marco, E-mail: fragai@cerm.unifi.it [University of Florence, Magnetic Resonance Center (CERM) (Italy)

    2016-04-15

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in {sup 15}N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  15. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.

    Science.gov (United States)

    Aricescu, A Radu; Owens, Raymond J

    2013-06-01

    Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.

    Science.gov (United States)

    Lamartina, S; Roscilli, G; Rinaudo, D; Delmastro, P; Toniatti, C

    1998-09-01

    Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

  17. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    Science.gov (United States)

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Nitrosothiol signaling and protein nitrosation in cell death.

    Science.gov (United States)

    Iyer, Anand Krishnan V; Rojanasakul, Yon; Azad, Neelam

    2014-11-15

    Nitric oxide, a reactive free radical, is an important signaling molecule that can lead to a plethora of cellular effects affecting homeostasis. A well-established mechanism by which NO manifests its effect on cellular functions is the post-translational chemical modification of cysteine thiols in substrate proteins by a process known as S-nitrosation. Studies that investigate regulation of cellular functions through NO have increasingly established S-nitrosation as the primary modulatory mechanism in their respective systems. There has been a substantial increase in the number of reports citing various candidate proteins undergoing S-nitrosation, which affects cell-death and -survival pathways in a number of tissues including heart, lung, brain and blood. With an exponentially growing list of proteins being identified as substrates for S-nitrosation, it is important to assimilate this information in different cell/tissue systems in order to gain an overall view of protein regulation of both individual proteins and a class of protein substrates. This will allow for broad mapping of proteins as a function of S-nitrosation, and help delineate their global effects on pathophysiological responses including cell death and survival. This information will not only provide a much better understanding of overall functional relevance of NO in the context of various disease states, it will also facilitate the generation of novel therapeutics to combat specific diseases that are driven by NO-mediated S-nitrosation. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    Science.gov (United States)

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  20. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  1. A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

    Science.gov (United States)

    Wang, Mingyu; Zhao, Qiushuang; Yang, Jinghua; Jiang, Baojie; Wang, Fangzhong; Liu, Kuimei; Fang, Xu

    2013-01-01

    The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei

  2. A mitogen-activated protein kinase Tmk3 participates in high osmolarity resistance, cell wall integrity maintenance and cellulase production regulation in Trichoderma reesei.

    Directory of Open Access Journals (Sweden)

    Mingyu Wang

    Full Text Available The mitogen-activated protein kinase (MAPK pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, 'budded' hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest

  3. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  4. Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid

    Directory of Open Access Journals (Sweden)

    Christopher J. Petzold

    2013-10-01

    Full Text Available Cell density is the critical parameter controlling tendon morphogenesis. Knowing its neighbors allows a cell to regulate correctly its proliferation and collagen production. A missing link to understanding this process is a molecular description of the sensing mechanism. Previously, this mechanism was shown in cell culture to rely on a diffusible factor (SNZR [sensor] with an affinity for the cell layer. This led to purifying conditioned medium over 4 columns and analyzing the final column fractions for band intensity on SDS gels versus biological activity – a 16 kD band strongly correlated between assays. N-terminal sequencing – EPLAVVDL – identified a large gene (424 AA, extremely conserved between chicken and human. In this paper we probe whether this is the correct gene. Can the predicted large protein be cleaved to a smaller protein? EPLAVVDL occurs towards the C-terminus and cleavage would create a small 94 AA protein. This protein would run at ∼10 kD, so what modifications or cofactor binding accounts for its running at 16 kD on SDS gels? This protein has no prominent hydrophobic regions, so can it be secreted? To validate its role, the chicken cDNA for this gene was tagged with myc and his and transfected into a human osteosarcoma cell line (U2OS. U2OS cells expressed the gene but not passively: differentiating into structures resembling spongy bone and expressing alkaline phosphatase, an early bone marker. Intracellularly, two bands were observed by Western blotting: the full length protein and a smaller form (26 kD. Outside the cell, a small band (28 kD was detected, although it was 40% larger than expected, as well as multiple larger bands. These larger forms could be converted to the predicted smaller protein (94 AA + tags by changing salt concentrations and ultrafiltering – releasing a cofactor to the filtrate while leaving a protein factor in the retentate. Using specific degradative enzymes and mass spectrometry, the

  5. The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein

    NARCIS (Netherlands)

    Giepmans, B N; Moolenaar, W H

    1998-01-01

    Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated

  6. Different Cells Make Different Proteins: A Laboratory Exercise Illustrating Tissue-Specific Protein Expression in Animals

    Science.gov (United States)

    Ibarguren, Izaskun; Villamarín, Antonio

    2017-01-01

    All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to…

  7. Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata.

    Science.gov (United States)

    Balachandran, S; Xiang, Y; Schobert, C; Thompson, G A; Lucas, W J

    1997-12-09

    In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20-100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

  8. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

    Directory of Open Access Journals (Sweden)

    Dawid Krokowski

    2017-12-01

    Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

  9. Dr. PIAS: an integrative system for assessing the druggability of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Furuya Toshio

    2011-02-01

    Full Text Available Abstract Background The amount of data on protein-protein interactions (PPIs available in public databases and in the literature has rapidly expanded in recent years. PPI data can provide useful information for researchers in pharmacology and medicine as well as those in interactome studies. There is urgent need for a novel methodology or software allowing the efficient utilization of PPI data in pharmacology and medicine. Results To address this need, we have developed the 'Druggable Protein-protein Interaction Assessment System' (Dr. PIAS. Dr. PIAS has a meta-database that stores various types of information (tertiary structures, drugs/chemicals, and biological functions associated with PPIs retrieved from public sources. By integrating this information, Dr. PIAS assesses whether a PPI is druggable as a target for small chemical ligands by using a supervised machine-learning method, support vector machine (SVM. Dr. PIAS holds not only known druggable PPIs but also all PPIs of human, mouse, rat, and human immunodeficiency virus (HIV proteins identified to date. Conclusions The design concept of Dr. PIAS is distinct from other published PPI databases in that it focuses on selecting the PPIs most likely to make good drug targets, rather than merely collecting PPI data.

  10. Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

    2012-01-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

  11. Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

    Science.gov (United States)

    Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

    2012-11-01

    The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

  12. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

    Science.gov (United States)

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

  13. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  14. Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

    Science.gov (United States)

    Suwandittakul, Nantana; Reamtong, Onrapak; Molee, Pattamaporn; Maneewatchararangsri, Santi; Sutherat, Maleerat; Chaisri, Urai; Wongkham, Sopit; Adisakwattana, Poom

    2017-09-07

    Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

  15. RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 2

    International Nuclear Information System (INIS)

    Skog, S.; Tribukait, B.; Nygard, O.; Wenner-Gren-Center foer Vetenskaplig Forskning, Stockholm

    1985-01-01

    Poly(A)-containing RNA (m-RNA) was studied in in vivo growing Ehrlich ascites tumour cells following a roentgen irradiation dose of 5 Gy. m-RNA increased significantly during the first 12 hours after irradiation. Thus, the observed decrease in protein synthesis rate during this time seems not to be due to radiation induced changes at the transcriptional level. The protein synthesis rate of in vivo irradiated cells incubated in vitro in culture medium was unchanged. On the other hand, the protein synthesis rate of non-irradiated cells incubated in vitro in ascites fluid from irradiated animals was decreased. We concluded that factor(s) inhibiting protein synthesis or the lack of factor(s) promoting protein synthesis in the ascites fluid is(are) of significance for the reduced protein synthesis of tumour cells found in irradiated in vivo growing cells. (orig.)

  16. The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    Directory of Open Access Journals (Sweden)

    María F. Organista

    2012-10-01

    The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.

  17. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

    International Nuclear Information System (INIS)

    Wang, Y.-H.; Chen, C.-P.; Chan, M.-H.; Chang, M.; Hou, Y.-W.; Chen, H.-H.; Hsu, H.-R.; Liu, Kevin; Lee, H.-J.

    2006-01-01

    Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or β-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo

  19. Periarteriolar Glioblastoma Stem Cell Niches Express Bone Marrow Hematopoietic Stem Cell Niche Proteins

    NARCIS (Netherlands)

    Hira, Vashendriya V. V.; Wormer, Jill R.; Kakar, Hala; Breznik, Barbara; van der Swaan, Britt; Hulsbos, Renske; Tigchelaar, Wikky; Tonar, Zbynek; Khurshed, Mohammed; Molenaar, Remco J.; van Noorden, Cornelis J. F.

    2018-01-01

    In glioblastoma, a fraction of malignant cells consists of therapy-resistant glioblastoma stem cells (GSCs) residing in protective niches that recapitulate hematopoietic stem cell (HSC) niches in bone marrow. We have previously shown that HSC niche proteins stromal cell-derived factor-1α (SDF-1α),

  20. Intracellular proteins produced by mammalian cells in response to environmental stress

    Science.gov (United States)

    Goochee, Charles F.; Passini, Cheryl A.

    1988-01-01

    The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

  1. Dissecting functions of the retinoblastoma tumor suppressor and the related pocket proteins by integrating genetic, cell biology, and electrophoretic techniques

    DEFF Research Database (Denmark)

    Hansen, Klaus; Lukas, J; Holm, K

    1999-01-01

    The members of the 'pocket protein' family, comprising the retinoblastoma tumor suppressor (pRB) and its relatives, p107 and p130, negatively regulate cell proliferation and modulate fundamental biological processes including embryonic development, differentiation, homeostatic tissue renewal...

  2. Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

    Science.gov (United States)

    Balachandran, Suchandra; Xiang, Yu; Schobert, Christian; Thompson, Gary A.; Lucas, William J.

    1997-01-01

    In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules. PMID:9391168

  3. RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. Pt. 1

    International Nuclear Information System (INIS)

    Skog, S.; Tribukait, B.; Sundius, G.

    1985-01-01

    The effects of roentgen irradiation on the incorporation of 3 H-uridine and 14 C-leucine into RNA and protein and the RNA and protein contents of in vivo growing Ehrlich ascites tumour cells were studied. The results were related to changes in the composition of cells in cell cycle and compared with the synthesis of RNA and protein in cell material from various parts of the cell cycle obtained by means of elutriator centrifuging. The incorporation expressed by the ratio between acid insoluble/acid soluble activity was unchanged for RNA during the observation period up to 24 hours after a dose of 5.0 Gy. The ratio for protein was markedly decreased between 4 and 24 hours. This decrease was partly due to a decrease of the pool size of leucine as studied by changing the amounts of 14 C leucine used. From these studies, the existence of at least two pools, an expandable and a non-expandable fixed pool can be concluded. There were no differences in the decrease of protein-synthesis between cells from the various parts of the cell cycle. The RNA and protein contents of the irradiated cells from various parts of the cell cycle corresponded to those of non-irradiated cells except for G 1 /early S-phase cells at 15 and 24 hours after irradiation. Possible reasons for this discrepancy are discussed. (orig.)

  4. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

    Science.gov (United States)

    Roskoski, Robert

    2005-11-11

    Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

  5. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    DEFF Research Database (Denmark)

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

    2009-01-01

    Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

  6. Light-induced protein degradation in human-derived cells.

    Science.gov (United States)

    Sun, Wansheng; Zhang, Wenyao; Zhang, Chao; Mao, Miaowei; Zhao, Yuzheng; Chen, Xianjun; Yang, Yi

    2017-05-27

    Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides.

    Science.gov (United States)

    Bolhassani, Azam; Jafarzade, Behnaz Sadat; Mardani, Golnaz

    2017-01-01

    The failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Cobos Everardo

    2005-01-01

    Full Text Available Abstract Background Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. Results One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20 with three members (FAM20A, FAM20B and FAM20C in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. Conclusions The FAM20 family represents a new family of secreted proteins with potential functions in regulating

  9. Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

    Directory of Open Access Journals (Sweden)

    Anantha Koteswararao Kanugula

    Full Text Available Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.

  10. Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

    Science.gov (United States)

    Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

    2017-06-01

    We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

  11. Targeting tumor-initiating cells: Eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction

    Science.gov (United States)

    Lamb, Rebecca; Harrison, Hannah; Smith, Duncan L.; Townsend, Paul A.; Jackson, Thomas; Ozsvari, Bela; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

    2015-01-01

    We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than “bulk” cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to

  12. Biological Evaluation of Single Cell Protein

    International Nuclear Information System (INIS)

    Hasan, I.A.; Mohamed, N.E.; El-Sayed, E.A.; Younis, N.A.

    2011-01-01

    In this study, the nutritional value of single cell protein (SCP) was evaluated as a non conventional protein source produced by fermenting fungal local strains of Trichoderma longibrachiatum, Aspergillus niger, Aspergillus terreus and Penicillium funiculosum with alkali treated sugar cane bagasse. Amino acid analysis revealed that the produced SCP contains essential and non essential amino acids. Male mice were fed on normal (basal) diet which contains 18% conventional protein and served as control group. In the second (T1) and the third (T2) group, the animals were fed on a diet in which 15% and 30% of conventional protein source were replaced by SCP, respectively. At intervals of 15, 30, 45 and 60 days, mice were sacrificed and the blood samples were collected for the biochemical evaluation. The daily averages of body weight were significantly higher with group T2 than group T1. Where as, the kidney weights in groups (T1) and (T2) were significantly increased as compared with control. A non significant difference between the tested groups in the enzyme activities of AST, ALT and GSH content of liver tissue were recorded. While, cholesterol and triglycerides contents showed a significant decrease in both (T1) and (T2) groups as compared with control. The recorded values of the serum hormone (T4), ALP activities, albumin and A/G ratio did not changed by the previous treatments. Serum levels of total protein, urea, creatinine and uric acid were higher for groups (T1) and (T2) than the control group. In conclusion, partial substitution of soy bean protein in mice diet with single cell protein (15%) improved the mice growth without any adverse effects on some of the physiological functions tested

  13. Identification of novel type 1 diabetes candidate genes by integrating genome-wide association data, protein-protein interactions, and human pancreatic islet gene expression

    DEFF Research Database (Denmark)

    Bergholdt, Regine; Brorsson, Caroline; Palleja, Albert

    2012-01-01

    Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with dis......-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.......Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated...... with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize...

  14. BAG3: a multifaceted protein that regulates major cell pathways

    Science.gov (United States)

    Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

    2011-01-01

    Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

  15. Visualization and targeted disruption of protein interactions in living cells

    Science.gov (United States)

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  16. Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Félix E

    2008-01-01

    regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Δ arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Δ strains.

  17. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures.

    Science.gov (United States)

    Desgagné-Penix, Isabel; Khan, Morgan F; Schriemer, David C; Cram, Dustin; Nowak, Jacek; Facchini, Peter J

    2010-11-18

    Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates

  18. Technology advancement for integrative stem cell analyses.

    Science.gov (United States)

    Jeong, Yoon; Choi, Jonghoon; Lee, Kwan Hyi

    2014-12-01

    Scientists have endeavored to use stem cells for a variety of applications ranging from basic science research to translational medicine. Population-based characterization of such stem cells, while providing an important foundation to further development, often disregard the heterogeneity inherent among individual constituents within a given population. The population-based analysis and characterization of stem cells and the problems associated with such a blanket approach only underscore the need for the development of new analytical technology. In this article, we review current stem cell analytical technologies, along with the advantages and disadvantages of each, followed by applications of these technologies in the field of stem cells. Furthermore, while recent advances in micro/nano technology have led to a growth in the stem cell analytical field, underlying architectural concepts allow only for a vertical analytical approach, in which different desirable parameters are obtained from multiple individual experiments and there are many technical challenges that limit vertically integrated analytical tools. Therefore, we propose--by introducing a concept of vertical and horizontal approach--that there is the need of adequate methods to the integration of information, such that multiple descriptive parameters from a stem cell can be obtained from a single experiment.

  19. Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

    Science.gov (United States)

    Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

    2017-12-01

    Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

    Science.gov (United States)

    Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

    2017-07-01

    Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Detecting Protein-Protein Interactions in the Intact Cell of Bacillus subtilis (ATCC 6633)

    OpenAIRE

    Winters, Michael S.; Day, R. A.

    2003-01-01

    The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C2N2) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques a...

  2. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    International Nuclear Information System (INIS)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  3. Challenges in the Development of Functional Assays of Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Sophie Demarche

    2012-11-01

    Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  4. Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

    Science.gov (United States)

    Paulmurugan, Ramasamy; Gambhir, Sanjiv S

    2005-08-15

    Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

  5. RNA-seq based expression analysis of the CHO cell protein secretion pathway

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Kildegaard, Helene Faustrup

    The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Although CHO cells have been used for more than 25 years, the genome sequence was first published in 2011. So far there have been limited studies...... of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly understood cellular processes that control and limit recombinant protein production and secretion, a system-wide study was initiated to identify possible engineering targets relevant for therapeutic protein...

  6. Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

    Science.gov (United States)

    Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

    2012-09-25

    Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

  7. Cell-free protein synthesis for structure determination by X-ray crystallography.

    Science.gov (United States)

    Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

    2010-01-01

    Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

  8. Noise genetics: inferring protein function by correlating phenotype with protein levels and localization in individual human cells.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    2014-03-01

    Full Text Available To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.

  9. Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

    Science.gov (United States)

    Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

    2018-06-01

    Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Cocompartmentation of proteins and K+ within the living cell

    International Nuclear Information System (INIS)

    Kellermayer, M.; Ludany, A.; Jobst, K.; Szucs, G.; Trombitas, K.; Hazlewood, C.F.

    1986-01-01

    Monolayer H-50 tissue culture cells were treated with Triton X-100 and Brij 58 nonionic detergents, and their electron microscopic morphology along with the release of the intracellular proteins [ 35 S]methionine-labelled and 42 K-labelled K + were studied. Although Triton X-100 was more effective, both detergents removed the lipoid membranes within 5 min. The mobilization and solubilization of the cytoplasmic and nuclear proteins occurred much faster with Triton X-100 than with Brij 58. In Triton X-100-treated cells, the loss of K + was complete within 2 min. The loss of K + from the Brij 58-treated cells was complete only after 10 min and the mobilization of K + showed sigmoid-type release kinetics. These results support the view that most of K + and diffusible proteins are not freely dissolved in the cellular water, but they are cocompartmentalized inside the living cell

  11. Two endogenous proteins that induce cell wall extension in plants

    Science.gov (United States)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  12. Dielectrophoretic focusing integrated pulsed laser activated cell sorting

    Science.gov (United States)

    Zhu, Xiongfeng; Kung, Yu-Chun; Wu, Ting-Hsiang; Teitell, Michael A.; Chiou, Pei-Yu

    2017-08-01

    We present a pulsed laser activated cell sorter (PLACS) integrated with novel sheathless size-independent dielectrophoretic (DEP) focusing. Microfluidic fluorescence activated cell sorting (μFACS) systems aim to provide a fully enclosed environment for sterile cell sorting and integration with upstream and downstream microfluidic modules. Among them, PLACS has shown a great potential in achieving comparable performance to commercial aerosol-based FACS (>90% purity at 25,000 cells sec-1). However conventional sheath flow focusing method suffers a severe sample dilution issue. Here we demonstrate a novel dielectrophoresis-integrated pulsed laser activated cell sorter (DEP-PLACS). It consists of a microfluidic channel with 3D electrodes laid out to provide a tunnel-shaped electric field profile along a 4cmlong channel for sheathlessly focusing microparticles/cells into a single stream in high-speed microfluidic flows. All focused particles pass through the fluorescence detection zone along the same streamline regardless of their sizes and types. Upon detection of target fluorescent particles, a nanosecond laser pulse is triggered and focused in a neighboring channel to generate a rapidly expanding cavitation bubble for precise sorting. DEP-PLACS has achieved a sorting purity of 91% for polystyrene beads at a throughput of 1,500 particle/sec.

  13. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  14. Coal Integrated Gasification Fuel Cell System Study

    Energy Technology Data Exchange (ETDEWEB)

    Gregory Wotzak; Chellappa Balan; Faress Rahman; Nguyen Minh

    2003-08-01

    The pre-baseline configuration for an Integrated Gasification Fuel Cell (IGFC) system has been developed. This case uses current gasification, clean-up, gas turbine, and bottoming cycle technologies together with projected large planar Solid Oxide Fuel Cell (SOFC) technology. This pre-baseline case will be used as a basis for identifying the critical factors impacting system performance and the major technical challenges in implementing such systems. Top-level system requirements were used as the criteria to evaluate and down select alternative sub-systems. The top choice subsystems were subsequently integrated to form the pre-baseline case. The down-selected pre-baseline case includes a British Gas Lurgi (BGL) gasification and cleanup sub-system integrated with a GE Power Systems 6FA+e gas turbine and the Hybrid Power Generation Systems planar Solid Oxide Fuel Cell (SOFC) sub-system. The overall efficiency of this system is estimated to be 43.0%. The system efficiency of the pre-baseline system provides a benchmark level for further optimization efforts in this program.

  15. The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development.

    Science.gov (United States)

    Miyazaki, Masaki; Miyazaki, Kazuko; Chen, Kenian; Jin, Yi; Turner, Jacob; Moore, Amanda J; Saito, Rintaro; Yoshida, Kenichi; Ogawa, Seishi; Rodewald, Hans-Reimer; Lin, Yin C; Kawamoto, Hiroshi; Murre, Cornelis

    2017-05-16

    Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Covalent microcontact printing of proteins fro cell patterning

    NARCIS (Netherlands)

    Rozkiewicz, D.I.; Kraan, Yvonne M.; Werten, Marc W.T.; de Wolf, Frits A.; Subramaniam, Vinod; Ravoo, B.J.; Reinhoudt, David

    2006-01-01

    We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An

  17. Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.

    Science.gov (United States)

    Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L; Wu, Hui; Kitten, Todd

    2009-11-01

    Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified-a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (approximately 2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention.

  18. Alterations of proteins in MDCK cells during acute potassium deficiency.

    Science.gov (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

    2016-06-01

    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen

    2005-01-01

    in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...

  20. Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

    Science.gov (United States)

    Hu, Kunpeng; Huang, Pinzhu; Luo, Hui; Yao, Zhicheng; Wang, Qingliang; Xiong, Zhiyong; Lin, Jizong; Huang, He; Xu, Shilei; Zhang, Peng; Liu, Bo

    2017-08-01

    Mammalian-enabled (MENA) protein is an actin-regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)-like phenotypes in HCC cells. Real-time-PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA-overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony-forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial-to-mesenchymal transition (EMT) markers, extracellular signal-regulated kinases (ERK) phosphorylation, and the level of β-catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β-catenin signaling pathways.

  1. Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

    Science.gov (United States)

    Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

    2013-01-01

    Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

  2. Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

    Science.gov (United States)

    Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

    2015-09-11

    Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

  3. Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

    International Nuclear Information System (INIS)

    Rebersek, Matej; Kanduser, Masa; Miklavcic, Damijan

    2011-01-01

    Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer. A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment. Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected. We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation

  4. Determination of the Physical Status (Episomal/Integral of HPV by qPCR in Esophageal Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Fariborz Soheili

    2017-03-01

    Full Text Available Background: In cervical cancer, the carcinogenic mechanism of human papillomavirus (HPV occurs through the integration of viral DNA into the host genome. This process initiates with a disruption in the E2 open reading frame (ORF of the viral genome. Disruption of E2 ORF results in an increased expression of the viral oncoproteins, E6 and E7, by removal of E2 suppression effect on their promoters. E6 and E7 interfere with the normal cell cycle by degrading the p53 and pRb tumor suppressor proteins, respectively. Objectives: The objective of this study was to determine the physical status (episomal/integral of HPV genome in esophageal squamous cell carcinoma (ESCC. Materials and Methods: The rate of copy numbers of E2 and E6 genes in HPV-18 and HPV-16 positive samples were analyzed by quantitative polymerase chain reaction (qPCR in order to assess the physical status (episomal/integral of HPV. DNA extracts from HeLa cell line were used as the positive control. Results: The E2 gene was detected in 1 sample, co-infected with HPV-16 and HPV-18. While, E6 gene was detected in all 11 HPV positive samples. The qPCR analysis showed the presence of integrated form of viral DNA in all HPV positive samples and only 1 mixed episomal-integrated form was detected. Conclusion: The presence of integrated forms of high risk HPV-16 and HPV-18 genomes might reflect a crucial process towards malignant transformation of ESCC.