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Sample records for cells integral proteins

  1. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion

    PubMed Central

    Li, Zhuo; Hung, Cher; Paterson, Reay G.; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J.; Lamb, Robert A.; He, Biao

    2015-01-01

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin–neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion. PMID:26392524

  2. Deducing protein function by forensic integrative cell biology.

    PubMed

    Earnshaw, William C

    2013-12-01

    Our ability to sequence genomes has provided us with near-complete lists of the proteins that compose cells, tissues, and organisms, but this is only the beginning of the process to discover the functions of cellular components. In the future, it's going to be crucial to develop computational analyses that can predict the biological functions of uncharacterised proteins. At the same time, we must not forget those fundamental experimental skills needed to confirm the predictions or send the analysts back to the drawing board to devise new ones. PMID:24358025

  3. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  4. The Multifunctions of WD40 Proteins in Genome Integrity and Cell Cycle Progression

    PubMed Central

    Zhang, Caiguo; Zhang, Fan

    2015-01-01

    Eukaryotic genome encodes numerous WD40 repeat proteins, which generally function as platforms of protein-protein interactions and are involved in numerous biological process, such as signal transduction, gene transcriptional regulation, protein modifications, cytoskeleton assembly, vesicular trafficking, DNA damage and repair, cell death and cell cycle progression. Among these diverse functions, genome integrity maintenance and cell cycle progression are extremely important as deregulation of them is clinically linked to uncontrolled proliferative diseases such as cancer. Thus, we mainly summarize and discuss the recent understanding of WD40 proteins and their molecular mechanisms linked to genome stability and cell cycle progression in this review, thereby demonstrating their pervasiveness and importance in cellular networks. PMID:25653723

  5. Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

    PubMed Central

    Jiang, Liwen; Rogers, John C.

    1998-01-01

    Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV. PMID:9832548

  6. Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

    PubMed Central

    Close, Dan M.; Ripp, Steven; Sayler, Gary S.

    2009-01-01

    Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed. PMID:22291559

  7. An integrated cell-free metabolic platform for protein production and synthetic biology

    PubMed Central

    Jewett, Michael C; Calhoun, Kara A; Voloshin, Alexei; Wuu, Jessica J; Swartz, James R

    2008-01-01

    Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements. PMID:18854819

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

    SciTech Connect

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

    2010-06-15

    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 [{sup 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 [{sup 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-{kappa}B, 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.

  9. Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

    PubMed

    Narayanan, Aswathy; Pullepu, Dileep; Reddy, Praveen Kumar; Uddin, Wasim; Kabir, M Anaul

    2016-07-01

    The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors. PMID:26992923

  10. Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

    PubMed

    Sonar, S; Patel, N; Fischer, W; Rothschild, K J

    1993-12-21

    Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy. PMID:8268152

  11. The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity.

    PubMed

    Xu, Xilong; Xiong, Xiufang; Sun, Yi

    2016-07-01

    Ribosomal proteins (RPs), the essential components of the ribosome, are a family of RNA-binding proteins, which play prime roles in ribosome biogenesis and protein translation. Recent studies revealed that RPs have additional extra-ribosomal functions, independent of protein biosynthesis, in regulation of diverse cellular processes. Here, we review recent advances in our understanding of how RPs regulate apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both MDM2/p53-dependent and p53-independent mechanisms. We also discuss the roles of RPs in the maintenance of genome integrity via modulating DNA damage response and repair. We further discuss mutations or deletions at the somatic or germline levels of some RPs in human cancers as well as in patients of Diamond-Blackfan anemia and 5q- syndrome with high susceptibility to cancer development. Moreover, we discuss the potential clinical application, based upon abnormal levels of RPs, in biomarker development for early diagnosis and/or prognosis of certain human cancers. Finally, we discuss the pressing issues in the field as future perspectives for better understanding the roles of RPs in human cancers to eventually benefit human health. PMID:27294833

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

    PubMed

    Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

    2016-05-01

    Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. PMID:26994102

  13. JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

    PubMed

    Li, Yuxin; Wang, Xusheng; Cho, Ji-Hoon; Shaw, Timothy I; Wu, Zhiping; Bai, Bing; Wang, Hong; Zhou, Suiping; Beach, Thomas G; Wu, Gang; Zhang, Jinghui; Peng, Junmin

    2016-07-01

    Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration. PMID:27225868

  14. Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures.

    PubMed

    Vogel, Jens H; Anspach, Birger; Kroner, Karl-Heinz; Piret, James M; Haynes, Charles A

    2002-06-30

    Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration. PMID:12001173

  15. Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

    PubMed

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; Goto, Hisatsugu; Ledford, Julie G; Hsia, Bethany; Pastva, Amy M; Wright, Jo Rae

    2012-02-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation. PMID:22219327

  16. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    PubMed

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. PMID:26850366

  17. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

    PubMed Central

    Tang, Hongting; Wang, Shenghuan; Wang, Jiajing; Song, Meihui; Xu, Mengyang; Zhang, Mengying; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2016-01-01

    Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion. PMID:27156860

  18. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.

    PubMed

    Tang, Hongting; Wang, Shenghuan; Wang, Jiajing; Song, Meihui; Xu, Mengyang; Zhang, Mengying; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2016-01-01

    Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion. PMID:27156860

  19. Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo

    PubMed Central

    SUNDARAM, J.; KESHAVA, S.; GOPALAKRISHNAN, R.; ESMON, C. T.; PENDURTHI, U. R.; RAO, L . V. M.

    2014-01-01

    Summary Background Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. Objective: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. Methods Lipopolysaccharide (LPS)-induced vascular leakage in the lung and kidney, and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. Results Administration of FVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice. Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease-activated receptor 1 antagonist blocked the FVIIa-induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. Conclusions This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients. PMID:24977291

  20. Cutting Edge: Regulation of Exosome Secretion by the Integral MAL Protein in T Cells.

    PubMed

    Ventimiglia, Leandro N; Fernández-Martín, Laura; Martínez-Alonso, Emma; Antón, Olga M; Guerra, Milagros; Martínez-Menárguez, José Angel; Andrés, Germán; Alonso, Miguel A

    2015-08-01

    Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells. PMID:26109641

  1. A cell penetrating peptide-integrated and enediyne-energized fusion protein shows potent antitumor activity.

    PubMed

    Ru, Qin; Shang, Bo-Yang; Miao, Qing-Fang; Li, Liang; Wu, Shu-Ying; Gao, Rui-Juan; Zhen, Yong-Su

    2012-11-20

    Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs. PMID:22982402

  2. Simulation of an integrated system for the production of methane and single cell protein from biomass

    SciTech Connect

    Thomas, M.V.

    1989-01-01

    A numerical model was developed to simulate the operation of an integrated system for the production of methane and single-cell algal protein from a variety of biomass energy crops or waste streams. Economic analysis was performed at the end of each simulation. The model was capable of assisting in the determination of design parameters by providing relative economic information for various strategies. Three configurations of anaerobic reactors were simulated. These included fed-bed reactors, conventional stirred tank reactors, and continuously expanding reactors. A generic anaerobic digestion process model, using lumped substrate parameters, was developed for use by type-specific reactor models. The generic anaerobic digestion model provided a tool for the testing of conversion efficiencies and kinetic parameters for a wide range of substrate types and reactor designs. Dynamic growth models were used to model the growth of algae and Eichornia crassipes was modeled as a function of daily incident radiation and temperature. The growth of Eichornia crassipes was modeled for the production of biomass as a substrate for digestion. Computer simulations with the system model indicated that tropical or subtropical locations offered the most promise for a viable system. The availability of large quantities of digestible waste and low land prices were found to be desirable in order to take advantage of the economies of scale. Other simulations indicated that poultry and swine manure produced larger biogas yields than cattle manure. The model was created in a modular fashion to allow for testing of a wide variety of unit operations. Coding was performed in the Pascal language for use on personal computers.

  3. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    PubMed

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis. PMID:27113856

  4. Cbk1 regulation of the RNA binding protein Ssd1 integrates cell fate with translational control

    PubMed Central

    Jansen, Jaclyn M.; Wanless, Antony G.; Seidel, Christopher W.; Weiss, Eric L.

    2009-01-01

    Summary Spatial control of gene expression, at the level of both transcription and translation, is critical for cellular differentiation [1-4]. In budding yeast, the conserved Ndr/warts kinase Cbk1 localizes to the new daughter cell where it acts as a cell fate determinant. Cbk1 both induces a daughter-specific transcriptional program and promotes morphogenesis in a less well-defined role [5-8]. Cbk1 is essential in cells expressing functional Ssd1, an RNA binding protein of unknown function [9-11]. We show that Cbk1 inhibits Ssd1 in vivo. Loss of this regulation dramatically slows bud expansion, leading to highly aberrant cell wall organization at the site of cell growth. Ssd1 associates with specific mRNAs, a significant number of which encode cell wall remodeling proteins. Translation of these messages is rapidly and specifically suppressed when Cbk1 is inhibited; this suppression requires Ssd1. Transcription of several of these Ssd1-associated mRNAs is also regulated by Cbk1, indicating that the kinase controls both the transcription and translation of daughter-specific mRNAs. This work suggests a novel system by which cells coordinate localized expression of genes involved in processes critical for cell growth and division. PMID:19962308

  5. Candida albicans cell shaving uncovers new proteins involved in cell wall integrity, yeast to hypha transition, stress response and host-pathogen interaction

    PubMed Central

    Hernáez, María Luisa; Reales-Calderon, Jose Antonio; Solis, Norma V.; Filler, Scott G.; Monteoliva, Lucia; Gil, Concha

    2015-01-01

    The ability to switch from yeast to hyphal growth is essential for virulence in Candida albicans. The cell surface is the initial point of contact between the fungus and the host. In this work, a free-gel proteomic strategy based on tryptic digestion of live yeast and hyphae cells and protein identification using LC-MS/MS methodology was used to identify cell surface proteins. Using this strategy, a total of 943 proteins were identified, of which 438 were in yeast and 928 were in hyphae. Of these proteins, 79 were closely related to the organization and biogenesis of the cell wall, including 28 GPI-anchored proteins, such as Hyr1 and Sod5 which were detected exclusively in hyphae, and Als2 and Sap10which were detected only in yeast. A group of 17 proteins of unknown function were subsequently studied by analysis of the corresponding deletion mutants. We found that four new proteins, Pst3, Tos1, Orf19.3060 and Orf19.5352 are involved in cell wall integrity and in C. albicans’ engulfment by macrophages. Moreover, the putative NADH-ubiquinone-related proteins, Ali1, Mci4, Orf19.287 and Orf19.7590, are also involved in osmotic and oxidative resistance, yeast to hypha transition and the ability to damage and invade oral epithelial cells. PMID:26087349

  6. Zebrafish Class 1 Phosphatidylinositol Transfer Proteins: PITPβ and Double Cone Cell Outer Segment Integrity in Retina

    PubMed Central

    Ile, Kristina E.; Kassen, Sean; Cao, Canhong; Vihtehlic, Thomas; Shah, Sweety D.; Mousley, Carl J.; Alb, James G.; Huijbregts, Richard P.H.; Stearns, George W.; Brockerhoff, Susan E.; Hyde, David R.; Bankaitis, Vytas A.

    2010-01-01

    Phosphatidylinositol transfer proteins (PITPs) in yeast coordinate lipid metabolism with the activities of specific membrane trafficking pathways. The structurally unrelated metazoan-specific PITPs (mPITPs), on the other hand, are an under-investigated class of proteins. It remains unclear what biological activities mPITPs discharge, and the mechanisms by which these proteins function are also not understood. The soluble class 1 mPITPs include the PITPα and PITPβ isoforms. Of these, the β-isoforms are particularly poorly characterized. Herein, we report the use of zebrafish as a model vertebrate for the study of class 1 mPITP biological function. Zebrafish express PITPα and PITPβ-isoforms (Pitpna and Pitpnb, respectively) and a novel PITPβ-like isoform (Pitpng). Pitpnb expression is particularly robust in double cone cells of the zebrafish retina. Morpholino-mediated protein knockdown experiments demonstrate Pitpnb activity is primarily required for biogenesis/maintenance of the double cone photoreceptor cell outer segments in the developing retina. By contrast, Pitpna activity is essential for successful navigation of early developmental programs. This study reports the initial description of the zebrafish class 1 mPITP family, and the first analysis of PITPβ function in a vertebrate. PMID:20545905

  7. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    /or misfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  8. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    SciTech Connect

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    aggregation and/or mislfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  9. Role of Protein Glycosylation in Candida parapsilosis Cell Wall Integrity and Host Interaction

    PubMed Central

    Pérez-García, Luis A.; Csonka, Katalin; Flores-Carreón, Arturo; Estrada-Mata, Eine; Mellado-Mojica, Erika; Németh, Tibor; López-Ramírez, Luz A.; Toth, Renata; López, Mercedes G.; Vizler, Csaba; Marton, Annamaria; Tóth, Adél; Nosanchuk, Joshua D.; Gácser, Attila; Mora-Montes, Héctor M.

    2016-01-01

    Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, Och1 is a Golgi α1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by β-elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1β stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNFα and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1Δ null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans. PMID:27014229

  10. Effects of the tumor inhibitory triterpenoid avicin G on cell integrity, cytokinesis, and protein ubiquitination in fission yeast

    PubMed Central

    Gutterman, Jordan U.; Lai, Hong T.; Yang, Peirong; Haridas, Valsala; Gaikwad, Amos; Marcus, Stevan

    2005-01-01

    Avicins comprise a class of triterpenoid compounds that exhibit tumor inhibitory activity. Here we show that avicin G is inhibitory to growth of the fission yeast Schizosaccharomyces pombe. S. pombe cells treated with a lethal concentration of avicin G (20 μM) exhibited a shrunken morphology, indicating that avicin G adversely affects cell integrity. Cells treated with a sublethal concentration of avicin G (6.5 μM) exhibited a strong cytokinesis-defective phenotype (multiseptated cells), as well as cell morphology defects. These phenotypes bear resemblance to those resulting from loss of Rho1 GTPase function in S. pombe. Indeed, Rho1-deficient S. pombe cells were strongly hypersensitive to avicin G, suggesting that the compound may perturb Rho1-dependent processes. Consistent with previously observed effects in human Jurkat T cells, avicin G treatment resulted in hyperaccumulation of ubiquitinated proteins in S. pombe cells. Interestingly, proteasome-defective S. pombe mutants were not markedly hypersensitive to avicin G, whereas an anaphase-promoting complex (mitotic ubiquitin ligase) mutant exhibited avicin G resistance, suggesting that the increase in levels of ubiquitinated proteins resulting from avicin G treatment may be due to increased protein ubiquitination, rather than inhibition of 26S proteasome activity. Mutants defective in the cAMP/PKA pathway also exhibited resistance to avicin G. Our results suggest that S. pombe will be a useful model organism for elucidating molecular targets of avicin G and serve as a guide to clinical application where dysfunctional aspects of Rho and/or ubiquitination function have been demonstrated as in cancer, fibrosis, and inflammation. PMID:16118282

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

    PubMed

    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

    2015-06-01

    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

  12. HNdb: an integrated database of gene and protein information on head and neck squamous cell carcinoma

    PubMed Central

    Henrique, Tiago; José Freitas da Silveira, Nelson; Henrique Cunha Volpato, Arthur; Mioto, Mayra Mataruco; Carolina Buzzo Stefanini, Ana; Bachir Fares, Adil; Gustavo da Silva Castro Andrade, João; Masson, Carolina; Verónica Mendoza López, Rossana; Daumas Nunes, Fabio; Paulo Kowalski, Luis; Severino, Patricia; Tajara, Eloiza Helena

    2016-01-01

    The total amount of scientific literature has grown rapidly in recent years. Specifically, there are several million citations in the field of cancer. This makes it difficult, if not impossible, to manually retrieve relevant information on the mechanisms that govern tumor behavior or the neoplastic process. Furthermore, cancer is a complex disease or, more accurately, a set of diseases. The heterogeneity that permeates many tumors is particularly evident in head and neck (HN) cancer, one of the most common types of cancer worldwide. In this study, we present HNdb, a free database that aims to provide a unified and comprehensive resource of information on genes and proteins involved in HN squamous cell carcinoma, covering data on genomics, transcriptomics, proteomics, literature citations and also cross-references of external databases. Different literature searches of MEDLINE abstracts were performed using specific Medical Subject Headings (MeSH terms) for oral, oropharyngeal, hypopharyngeal and laryngeal squamous cell carcinomas. A curated gene-to-publication assignment yielded a total of 1370 genes related to HN cancer. The diversity of results allowed identifying novel and mostly unexplored gene associations, revealing, for example, that processes linked to response to steroid hormone stimulus are significantly enriched in genes related to HN carcinomas. Thus, our database expands the possibilities for gene networks investigation, providing potential hypothesis to be tested. Database URL: http://www.gencapo.famerp.br/hndb PMID:27013077

  13. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    PubMed

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  14. PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

    PubMed Central

    Teichert, Ines; Steffens, Eva Katharina; Schnaß, Nicole; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk A.; Kück, Ulrich

    2014-01-01

    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. PMID:25188365

  15. Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

    PubMed Central

    Richter, Uwe; Lahtinen, Taina; Marttinen, Paula; Suomi, Fumi

    2015-01-01

    Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes. PMID:26504172

  16. The Conserved Hypothetical Protein Rv0574c Is Required for Cell Wall Integrity, Stress Tolerance, and Virulence of Mycobacterium tuberculosis

    PubMed Central

    Garg, Rajni; Tripathi, Deeksha; Kant, Sashi; Chandra, Harish; Bhatnagar, Rakesh

    2014-01-01

    The virulence of Mycobacterium tuberculosis is intimately related to its distinctive cell wall. The biological significance of poly-α-l-glutamine (PLG), a component in the cell wall of virulent mycobacteria, has not been explored adequately. The focus of this study is to investigate the role of a locus, Rv0574c, coding for a polyglutamate synthase-like protein, in the synthesis of poly-α-l-glutamine in the context of mycobacterial virulence. Evaluation of Rv0574c gene expression in M. tuberculosis demonstrated its growth-phase-linked induction with concomitant accumulation of poly-α-l-glutamine in the cell wall. Rv0574c was activated under conditions prevalent in the tubercular granuloma, e.g., hypoxia, nitric oxide, and CO2. For functional characterization, we produced a deletion mutant of the Rv0574c gene by allelic exchange. The mutant produced smaller amounts of poly-α-l-glutamine in the cell wall than did the wild-type bacterium. Additionally, the increased sensitivity of the mutant to antitubercular drugs, SDS, lysozyme, and mechanical stress was accompanied by a drastic reduction in the ability to form biofilm. Growth of the ΔRv0574c strain was normal under in vitro conditions but was retarded in THP-1 macrophages and in the lungs and spleen of BALB/c mice. This was in agreement with histopathology of the lungs showing slow growth and less severe pathology than that of the wild-type strain. In summary, this study demonstrates that the protein encoded by the Rv0574c locus, by virtue of modulating PLG content in the cell wall, helps in maintaining cellular integrity in a hostile host environment. Also, its involvement in protecting the pathogen from host-generated lethal factors contributes to the infectious biology of M. tuberculosis. PMID:25312955

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Integrated circuit cell library

    NASA Technical Reports Server (NTRS)

    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.

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

    ERIC Educational Resources Information Center

    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. Effect of mycobacterial secretory proteins on the cellular integrity and cytokine profile of type II alveolar epithelial cells

    PubMed Central

    Adlakha, Nidhi; Vir, Pooja; Verma, Indu

    2012-01-01

    Background: Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis (M. tb). In lungs, alveolar macrophages and type II alveolar epithelial cells serve as a replicative niche for this pathogen. Secretory proteins released by actively replicating tubercle bacilli are known to interact with host cells at the initial stages of infection. To understand the role of these cells in TB pathogenesis, it is important to identify the mycobacterial components involved in interaction with alveolar epithelial cells. Materials and Methods: We fractionated the whole secretory proteome of M. tb H37Rv into 10 narrow molecular mass fractions (A1-A10; <20 kDa to >90 kDa) that were studied for their binding potential with A549; type II alveolar epithelial cell line. We also studied the consequences of this interaction in terms of change in epithelial cell viability by MTT assay and cytokine release by ELISA. Results: Our results show that several mycobacterial proteins bind and confer cytolysis in epithelial cells. Amongst all the fractions, proteins ranging from 35-45 kDa (A5) exhibited highest binding to A549 cells with a consequence of cytolysis of these cells. This fraction (A5) also led to release of various cytokines important in anti-mycobacterial immunity. Conclusion: Fraction A5 (35-45 kDa) of mycobacterial secretory proteome play an important role in mediating M. tb interaction with type II alveolar epithelial cells with the consequences detrimental for the TB pathogenesis. Further studies are being carried out to identify the candidate proteins from this region. PMID:23243342

  1. Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

    PubMed Central

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

    2015-01-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  2. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    PubMed

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  3. The Cell-Free Integration of a Polytopic Mitochondrial Membrane Protein into Liposomes Occurs Cotranslationally and in a Lipid-Dependent Manner

    PubMed Central

    Long, Ashley R.; O'Brien, Catherine C.; Alder, Nathan N.

    2012-01-01

    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. PMID:23050015

  4. Cell wall integrity

    PubMed Central

    Pogorelko, Gennady; Lionetti, Vincenzo; Bellincampi, Daniela; Zabotina, Olga

    2013-01-01

    The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways. PMID:23857352

  5. Integrated continuous production of recombinant therapeutic proteins.

    PubMed

    Warikoo, Veena; Godawat, Rahul; Brower, Kevin; Jain, Sujit; Cummings, Daniel; Simons, Elizabeth; Johnson, Timothy; Walther, Jason; Yu, Marcella; Wright, Benjamin; McLarty, Jean; Karey, Kenneth P; Hwang, Chris; Zhou, Weichang; Riske, Frank; Konstantinov, Konstantin

    2012-12-01

    In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. PMID:22729761

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

    PubMed Central

    Bondzio, Angelika; Aschenbach, Jörg R.; Bratz, Katharina; Einspanier, Ralf; Lodemann, Ulrike

    2015-01-01

    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. PMID:25948884

  7. Identification of putative negative regulators of yeast signaling through a screening for protein phosphatases acting on cell wall integrity and mating MAPK pathways.

    PubMed

    Sacristán-Reviriego, Almudena; Martín, Humberto; Molina, María

    2015-04-01

    The lack of signaling through MAPK pathways leads to a defective cellular response to the corresponding stimulus, but an improper hyperactivation of these routes results in deleterious effects as well. Protein phosphorylation is an activating modification for signal transmission through components of MAPK pathways and thus, protein phosphatases are key negative regulators of these cellular routes by limiting excessive signaling activity. However, in contrast to most of the protein kinases operating in MAPK pathways, protein phosphatases usually exhibit redundancy and promiscuity, which has limited the identification of their function. In order to identify new putative phosphatases operating in Saccharomyces cerevisiae MAPK signaling, we have taken advantage of growth inhibition promoted by overproduction of constitutively active components of the mating and cell wall integrity (CWI) pathways to perform a screen with a collection of 43 protein phosphatases or phosphatase-regulatory proteins. The phosphatases able to alleviate the induced growth inhibition when overproduced were further studied by testing their capacity to downregulate expression of mating and CWI responsive promoters and the consequences of their removal on MAPK signaling. Epistasis analysis placed the Ser/Thr protein phosphatase Ppq1 as a regulator of the mating MAPK module downstream the MAPKKK Ste11. The dual specificity phosphatase Yvh1 was found to be important for the maintenance of cell wall integrity and appropriate signaling through the CWI pathway. Moreover, we have found that Ptc2 and Ptc4 bind to the CWI MAPK Slt2. Together with known phosphatases of the mating and CWI pathway, as Msg5 or Ptp2, other putative negative regulators of both pathways that came up in the screening were Ptc2, Oca2 and Ptp1. We show that Ptp1 physically interacts with Slt2 and the mating MAPK Fus3. Elimination of Ptp1 results in increased signaling through these pathways, suggesting that this tyrosine

  8. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

  9. Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

    PubMed Central

    Evans, DRH.; Stark, MJR.

    1997-01-01

    Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene. PMID:9071579

  10. Investigation of the Biophysical and Cell Biological Properties of Ferroportin, a Multi-Pass Integral Membrane Protein Iron Exporter

    PubMed Central

    Rice, Adrian E.; Mendez, Michael J.; Hokanson, Craig A.; Rees, Douglas C.; Björkman, Pamela J.

    2009-01-01

    Ferroportin is a multi-pass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload disease hereditary hemochromatosis. Relatively little is known about ferroportin’s properties or the mechanism by which mutations cause disease. Here we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We show that purified, detergent-solubilized ferroportin was a well-folded monomer that bound hepcidin. In cell membranes, the N- and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all formed well-folded monomers that localized to the plasma membrane, but some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin’s role in regulating iron homeostasis in health and disease can be interpreted. PMID:19150361

  11. Integrative Proteomics and Tissue Microarray Profiling Indicate the Association between Overexpressed Serum Proteins and Non-Small Cell Lung Cancer

    PubMed Central

    Hu, Haichuan; Wang, Rui; Sun, Yihua; Zeng, Rong; Chen, Haiquan

    2012-01-01

    Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases. PMID:23284758

  12. The multi-facet aspects of cell sentience and their relevance for the integrative brain actions: role of membrane protein energy landscape.

    PubMed

    Agnati, Luigi F; Marcoli, Manuela; Maura, Guido; Fuxe, Kjell; Guidolin, Diego

    2016-06-01

    Several ion channels can be randomly and spontaneously in an open state, allowing the exchange of ion fluxes between extracellular and intracellular environments. We propose that the random changes in the state of ion channels could be also due to proteins exploring their energy landscapes. Indeed, proteins can modify their steric conformation under the effects of the physicochemical parameters of the environments with which they are in contact, namely, the extracellular, intramembrane and intracellular environments. In particular, it is proposed that the random walk of proteins in their energy landscape is towards attractors that can favor the open or close condition of the ion channels and/or intrinsic activity of G-protein-coupled receptors. The main aspect of the present proposal is that some relevant physicochemical parameters of the environments (e.g. molecular composition, temperature, electrical fields) with which some signaling-involved plasma membrane proteins are in contact alter their conformations. In turn, these changes can modify their information handling via a modulatory action on their random walk towards suitable attractors of their energy landscape. Thus, spontaneous and/or signal-triggered electrical activities of neurons occur that can have emergent properties capable of influencing the integrative actions of brain networks. Against this background, Cook's hypothesis on 'cell sentience' is developed by proposing that physicochemical parameters of the environments with which the plasma-membrane proteins of complex cellular networks are in contact fulfill a fundamental role in their spontaneous and/or signal-triggered activity. Furthermore, it is proposed that a specialized organelle, the primary cilium, which is present in most cells (also neurons and astrocytes), could be of peculiar importance to pick up chemical signals such as ions and transmitters and to detect physical signals such as pressure waves, thermal gradients, and local field

  13. Two Golgi integral membrane proteins (GIMPS) exhibit region- and cell type-specific distribution in the epididymis of the adult rat.

    PubMed

    Suarez-Quian, C A; Jelesoff, N

    1994-12-15

    The epididymis participates in the post-testicular maturation and storage of spermatozoa by secreting proteins into the tubule lumen in a region-specific fashion. The underlying molecular mechanisms leading to biogenesis of these region-specific differences, however, are not known, although components of the Golgi complex membrane container must undoubtedly be intimately involved. Two monoclonal antibodies raised against Golgi integral membrane proteins, recognizing either the cis (GIMPc) or trans Golgi (GIMPt) cisternae, were used as molecular probes of these regions to begin the characterization of the Golgi complex of in vivo and in vitro epididymal cells. Immunolocalization of GIMPs was performed on frozen sections and in cultured cells using biotin-streptavidin-peroxidase immunocytochemistry. In tissue sections, immunostaining of GIMPt was extremely robust in the supranuclear cytoplasm throughout the epididymis. In contrast, no GIMPc immunostaining was detected in the initial segment or in clear cells of the distal caput, corpus, and cauda. Immunodetection of GIMPc and GIMPt in epididymal cells in vitro revealed a reticular, perinuclear pattern, and NH4Cl treatment preferentially disrupted the GIMPt immunolocalization. These results characterizing the molecular components of the Golgi complex will form the basis of additional studies to gain further insight into mechanisms leading to generation of regional differences in epididymal function. PMID:7873795

  14. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

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

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4 % SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells. Graphical Abstract The legend of online abstract figure is (a) schematic illustration of membrane proteins extraction, purification and digestion from living cells; (b) diagrammatic sketch of the automatic integrated membrane proteome analysis system. PMID:26922343

  15. Bilayer-thickness-mediated interactions between integral membrane proteins.

    PubMed

    Kahraman, Osman; Koch, Peter D; Klug, William S; Haselwandter, Christoph A

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  16. Bilayer-thickness-mediated interactions between integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  17. The Saccharomyces cerevisiae 14-3-3 proteins are required for the G1/S transition, actin cytoskeleton organization and cell wall integrity.

    PubMed

    Lottersberger, Francisca; Panza, Andrea; Lucchini, Giovanna; Piatti, Simonetta; Longhese, Maria Pia

    2006-06-01

    14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G(1)/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G(1) cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G(1) transcriptional program. PMID:16648583

  18. Single-cell proteins

    SciTech Connect

    Litchfield, J.H.

    1983-02-11

    Both photosynthetic and nonphotosynthetic microorganisms, grown on various carbon and energy sources, are used in fermentation processes for the production of single-cell proteins. Commercial-scale production has been limited to two algal processes, one bacterial process, and several yeast and fungal processes. High capital and operating costs and the need for extensive nutritional and toxicological assessments have limited the development and commercialization of new processes. Any increase in commercial-scale production appears to be limited to those regions of the world where low-cost carbon and energy sources are available and conventional animal feedstuff proteins, such as soybean meal or fish meal, are in short supply. (Refs. 59).

  19. The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.

    PubMed

    Schmidt, A M; Mora, R; Cao, R; Yan, S D; Brett, J; Ramakrishnan, R; Tsang, T C; Simionescu, M; Stern, D

    1994-04-01

    Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane. PMID:8144581

  20. Regulation of age-related structural integrity in neurons by protein with tau-like repeats (PTL-1) is cell autonomous.

    PubMed

    Chew, Yee Lian; Fan, Xiaochen; Götz, Jürgen; Nicholas, Hannah R

    2014-01-01

    PTL-1 is the sole homolog of the MAP2/MAP4/tau family in Caenorhabditis elegans. Accumulation of tau is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease. Therefore, reducing tau levels has been suggested as a therapeutic strategy. We previously showed that PTL-1 maintains age-related structural integrity in neurons, implying that excessive reduction in the levels of a tau-like protein is detrimental. Here, we demonstrate that the regulation of neuronal ageing by PTL-1 occurs via a cell-autonomous mechanism. We re-expressed PTL-1 in a null mutant background using a pan-neuronal promoter to show that PTL-1 functions in neurons to maintain structural integrity. We next expressed PTL-1 only in touch neurons and showed rescue of the neuronal ageing phenotype of ptl-1 mutant animals in these neurons but not in another neuronal subset, the ventral nerve cord GABAergic neurons. Knockdown of PTL-1 in touch neurons also resulted in premature neuronal ageing in these neurons but not in GABAergic neurons. Additionally, expression of PTL-1 in touch neurons alone was unable to rescue the shortened lifespan observed in ptl-1 mutants, but pan-neuronal re-expression restored wild-type longevity, indicating that, at least for a specific group of mechanosensory neurons, premature neuronal ageing and organismal ageing can be decoupled. PMID:24898126

  1. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    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.

  2. FET proteins regulate lifespan and neuronal integrity

    PubMed Central

    Therrien, Martine; Rouleau, Guy A.; Dion, Patrick A.; Parker, J. Alex

    2016-01-01

    The FET protein family includes FUS, EWS and TAF15 proteins, all of which have been linked to amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motor neurons. Here, we show that a reduction of FET proteins in the nematode Caenorhabditis elegans causes synaptic dysfunction accompanied by impaired motor phenotypes. FET proteins are also involved in the regulation of lifespan and stress resistance, acting partially through the insulin/IGF-signalling pathway. We propose that FET proteins are involved in the maintenance of lifespan, cellular stress resistance and neuronal integrity. PMID:27117089

  3. MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae.

    PubMed

    Guo, Min; Gao, Fei; Zhu, Xiaolei; Nie, Xiang; Pan, YueMin; Gao, Zhimou

    2015-10-01

    The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae. PMID:26227409

  4. Chip integrated fuel cell accumulator

    NASA Astrophysics Data System (ADS)

    Frank, M.; Erdler, G.; Frerichs, H.-P.; Müller, C.; Reinecke, H.

    A unique new design of a chip integrated fuel cell accumulator is presented. The system combines an electrolyser and a self-breathing polymer electrolyte membrane (PEM) fuel cell with integrated palladium hydrogen storage on a silicon substrate. Outstanding advantages of this assembly are the fuel cell with integrated hydrogen storage, the possibility of refuelling it by electrolysis and the opportunity of simply refilling the electrolyte by adding water. By applying an electrical current, wiring the palladium hydrogen storage as cathode and the counter-electrode as anode, the electrolyser produces hydrogen at the palladium surface and oxygen at the electrolyser cell anode. The generated hydrogen is absorbed by the palladium electrode and the hydrogen storage is refilled consequently enabling the fuel cell to function.

  5. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

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

    PubMed

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    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 homolog in Colletotrichum 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. PMID:27536296

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

    PubMed Central

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    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 homolog in Colletotrichum 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. PMID:27536296

  8. Type 2C protein phosphatase Ptc6 participates in activation of the Slt2-mediated cell wall integrity pathway in Saccharomyces cerevisiae.

    PubMed

    Sharmin, Dilruba; Sasano, Yu; Sugiyama, Minetaka; Harashima, Satoshi

    2015-04-01

    The phosphorylation status of cellular proteins results from an equilibrium between the activities of protein kinases and protein phosphatases (PPases). Reversible protein phosphorylation is an important aspect of signal transduction that regulate many biological processes in eukaryotic cells. The Saccharomyces cerevisiae genome encodes 40 PPases, including seven members of the protein phosphatase 2C subfamily (PTC1 to PTC7). In contrast to other PPases, the cellular roles of PTCs have not been investigated in detail. Here, we sought to determine the cellular role of PTC6 in S. cerevisiae with disruption of PTC genes. We found that cells with Δptc6 disruption were tolerant to the cell wall-damaging agents Congo red (CR) and calcofluor white (CFW); however, cells with simultaneous disruption of PTC1 and PTC6 were very sensitive to these agents. Thus, simultaneous disruption of PTC1 and PTC6 gave a synergistic response to cell wall damaging agents. The level of phosphorylated Slt2 increased significantly after CR treatment in Δptc1 cells and more so in Δptc1Δptc6 cells; therefore, deletion of PTC6 enhanced Slt2 phosphorylation in the Δptc1 disruptant. The level of transcription of KDX1 upon exposure to CR increased to a greater extent in the Δptc1Δptc6 double disruptant than the Δptc1 single disruptant. The Δptc1Δptc6 double disruptant cells showed normal vacuole formation under standard growth conditions, but fragmented vacuoles were present in the presence of CR or CFW. Our analyses indicate that S. cerevisiae PTC6 participates in the negative regulation of Slt2 phosphorylation and vacuole morphogenesis under cell wall stress conditions. PMID:25449759

  9. An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells.

    PubMed Central

    Nugroho, T T; Mendenhall, M D

    1994-01-01

    The gene encoding a 40-kDa protein, previously studied as a substrate and inhibitor of the yeast cyclin-dependent protein kinase, Cdc28, has been cloned. The DNA sequence reveals that p40 is a highly charged protein of 32,187 Da with no significant homology to other proteins. Overexpression of the gene encoding p40, SIC1, produces cells with an elongated but morphology similar to that of cells with depleted levels of the CLB gene products, suggesting that p40 acts as an inhibitor of Cdc28-Clb complexes in vivo. A SIC1 deletion is viable and has highly increased frequencies of broken and lost chromosomes. The deletion strain segregates out many dead cells that are primarily arrested at the G2 checkpoint in an asymmetric fashion. Only daughters and young mothers display the lethal defect, while experienced mothers appear to grow normally. These results suggest that negative regulation of Cdc28 protein kinase activity by p40 is important for faithful segregation of chromosomes to daughter cells. Images PMID:8164683

  10. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  11. The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

    PubMed

    Celis, J E; Rasmussen, H H; Madsen, P; Leffers, H; Honoré, B; Dejgaard, K; Gesser, B; Olsen, E; Gromov, P; Hoffmann, H J

    1992-12-01

    The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease. PMID:1286666

  12. Integrative visual analysis of protein sequence mutations

    PubMed Central

    2014-01-01

    Background An important aspect of studying the relationship between protein sequence, structure and function is the molecular characterization of the effect of protein mutations. To understand the functional impact of amino acid changes, the multiple biological properties of protein residues have to be considered together. Results Here, we present a novel visual approach for analyzing residue mutations. It combines different biological visualizations and integrates them with molecular data derived from external resources. To show various aspects of the biological information on different scales, our approach includes one-dimensional sequence views, three-dimensional protein structure views and two-dimensional views of residue interaction networks as well as aggregated views. The views are linked tightly and synchronized to reduce the cognitive load of the user when switching between them. In particular, the protein mutations are mapped onto the views together with further functional and structural information. We also assess the impact of individual amino acid changes by the detailed analysis and visualization of the involved residue interactions. We demonstrate the effectiveness of our approach and the developed software on the data provided for the BioVis 2013 data contest. Conclusions Our visual approach and software greatly facilitate the integrative and interactive analysis of protein mutations based on complementary visualizations. The different data views offered to the user are enriched with information about molecular properties of amino acid residues and further biological knowledge. PMID:25237389

  13. Quality control of integral membrane proteins by assembly-dependent membrane integration.

    PubMed

    Feige, Matthias J; Hendershot, Linda M

    2013-08-01

    Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis. PMID:23932713

  14. Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity.

    PubMed

    Chenette, Devon M; Cadwallader, Adam B; Antwine, Tiffany L; Larkin, Lauren C; Wang, Jinhua; Olwin, Bradley B; Schneider, Robert J

    2016-08-01

    Following skeletal muscle injury, muscle stem cells (satellite cells) are activated, proliferate, and differentiate to form myofibers. We show that mRNA-decay protein AUF1 regulates satellite cell function through targeted degradation of specific mRNAs containing 3' AU-rich elements (AREs). auf1(-/-) mice undergo accelerated skeletal muscle wasting with age and impaired skeletal muscle repair following injury. Satellite cell mRNA analysis and regeneration studies demonstrate that auf1(-/-) satellite cell self-renewal is impaired due to increased stability and overexpression of ARE-mRNAs, including cell-autonomous overexpression of matrix metalloprotease MMP9. Secreted MMP9 degrades the skeletal muscle matrix, preventing satellite-cell-mediated regeneration and return to quiescence. Blocking MMP9 activity in auf1(-/-) mice restores skeletal muscle repair and maintenance of the satellite cell population. Control of ARE-mRNA decay by AUF1 represents a mechanism for adult stem cell regulation and is implicated in human skeletal muscle wasting diseases. PMID:27452471

  15. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.

    PubMed

    Marshall, Stephen S; Niesen, Michiel J M; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M; Galimidi, Rachel P; Zhang, Bin; Clemons, William M; Miller, Thomas F

    2016-08-23

    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

  16. A Link Between Integral Membrane Protein Expression and Simulated Integration Efficiency

    PubMed Central

    Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M.; Miller, Thomas F.

    2016-01-01

    Integral membrane proteins (IMP) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels widely vary and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

  17. The Biofilm Inhibitor Carolacton Disturbs Membrane Integrity and Cell Division of Streptococcus mutans through the Serine/Threonine Protein Kinase PknB ▿ †

    PubMed Central

    Reck, Michael; Rutz, Katrin; Kunze, Brigitte; Tomasch, Jürgen; Surapaneni, Subhash Kumar; Schulz, Stefan; Wagner-Döbler, Irene

    2011-01-01

    Carolacton, a secondary metabolite isolated from the myxobacterium Sorangium cellulosum, disturbs Streptococcus mutans biofilm viability at nanomolar concentrations. Here we show that carolacton causes leakage of cytoplasmic content (DNA and proteins) in growing cells at low pH and provide quantitative data on the membrane damage. Furthermore, we demonstrate that the biofilm-specific activity of carolacton is due to the strong acidification occurring during biofilm growth. The chemical conversion of the ketocarbonic function of the molecule to a carolacton methylester did not impact its activity, indicating that carolacton is not functionally activated at low pH by a change of its net charge. A comparative time series microarray analysis identified the VicKRX and ComDE two-component signal transduction systems and genes involved in cell wall metabolism as playing essential roles in the response to carolacton treatment. A sensitivity testing of mutants with deletions of all 13 viable histidine kinases and the serine/threonine protein kinase PknB of S. mutans identified only the ΔpknB deletion mutant as being insensitive to carolacton treatment. A strong overlap between the regulon of PknB in S. mutans and the genes affected by carolacton treatment was found. The data suggest that carolacton acts by interfering with PknB-mediated signaling in growing cells. The resulting altered cell wall morphology causes membrane damage and cell death at low pH. PMID:21840978

  18. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.

    PubMed

    Valmori, Danila; Souleimanian, Naira E; Tosello, Valeria; Bhardwaj, Nina; Adams, Sylvia; O'Neill, David; Pavlick, Anna; Escalon, Juliet B; Cruz, Crystal M; Angiulli, Angelica; Angiulli, Francesca; Mears, Gregory; Vogel, Susan M; Pan, Linda; Jungbluth, Achim A; Hoffmann, Eric W; Venhaus, Ralph; Ritter, Gerd; Old, Lloyd J; Ayyoub, Maha

    2007-05-22

    The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines. PMID:17517626

  19. Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

    PubMed Central

    Granato, Daniela C.; Zanetti, Mariana R.; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R.; Aragão, Annelize Z.; Agostini, Michelle; Carazzolle, Marcelo F.; Vidal, Ramon O.; Flores, Isadora L.; Korvala, Johanna; Cervigne, Nilva K.; Silva, Alan R. S.; Coletta, Ricardo D.; Graner, Edgard; Sherman, Nicholas E.; Leme, Adriana F. Paes

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. PMID:24858105

  20. Integrated transcriptomic and proteomic analysis identifies protein kinase CK2 as a key signaling node in an inflammatory cytokine network in ovarian cancer cells

    PubMed Central

    Kulbe, Hagen; Iorio, Francesco; Chakravarty, Probir; Milagre, Carla S.; Moore, Robert; Thompson, Richard G.; Everitt, Gemma; Canosa, Monica; Montoya, Alexander; Drygin, Denis; Braicu, Ioana; Sehouli, Jalid; Saez-Rodriguez, Julio; Cutillas, Pedro R.; Balkwill, Frances R.

    2016-01-01

    We previously showed how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in ovarian cancer. This network, which we named the “TNF network”, has paracrine actions within the tumor microenvironment, influencing angiogenesis and the immune cell infiltrate. The aim of this study was to identify critical regulators in the signaling pathways of the TNF network in ovarian cancer cells that might be therapeutic targets. To achieve our aim, we used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis. Among the potential therapeutic kinase targets identified was the protein kinase Casein kinase II (CK2). Knockdown of CK2 expression in malignant cells by siRNA or treatment with the specific CK2 inhibitor CX-4945 significantly decreased Notch signaling and reduced constitutive cytokine release in ovarian cancer cell lines that expressed the TNF network as well as malignant cells isolated from high grade serous ovarian cancer ascites. The expression of the same cytokines was also inhibited after treatment with CX-4945 in a 3D organotypic model. CK2 inhibition was associated with concomitant inhibition of proliferative activity, reduced angiogenesis and experimental peritoneal ovarian tumor growth. In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer. PMID:26871292

  1. The Pmt2p-Mediated Protein O-Mannosylation Is Required for Morphogenesis, Adhesive Properties, Cell Wall Integrity and Full Virulence of Magnaporthe oryzae

    PubMed Central

    Guo, Min; Tan, Leyong; Nie, Xiang; Zhu, Xiaolei; Pan, Yuemin; Gao, Zhimou

    2016-01-01

    Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT–PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae. PMID:27199956

  2. The Pmt2p-Mediated Protein O-Mannosylation Is Required for Morphogenesis, Adhesive Properties, Cell Wall Integrity and Full Virulence of Magnaporthe oryzae.

    PubMed

    Guo, Min; Tan, Leyong; Nie, Xiang; Zhu, Xiaolei; Pan, Yuemin; Gao, Zhimou

    2016-01-01

    Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT-PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae. PMID:27199956

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

    PubMed

    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. PMID:27322762

  4. Reprogramming cells with synthetic proteins.

    PubMed

    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. PMID:25652623

  5. Cell Stress Proteins in Atherothrombosis

    PubMed Central

    Madrigal-Matute, Julio; Martinez-Pinna, Roxana; Fernandez-Garcia, Carlos Ernesto; Ramos-Mozo, Priscila; Burillo, Elena; Egido, Jesus; Blanco-Colio, Luis Miguel; Martin-Ventura, Jose Luis

    2012-01-01

    Cell stress proteins (CSPs) are a large and heterogenous family of proteins, sharing two main characteristics: their levels and/or location are modified under stress and most of them can exert a chaperon function inside the cells. Nonetheless, they are also involved in the modulation of several mechanisms, both at the intracellular and the extracellular compartments. There are more than 100 proteins belonging to the CSPs family, among them the thioredoxin (TRX) system, which is the focus of the present paper. TRX system is composed of several proteins such as TRX and peroxiredoxin (PRDX), two thiol-containing enzymes that are key players in redox homeostasis due to their ability to scavenge potential harmful reactive oxygen species. In addition to their main role as antioxidants, recent data highlights their function in several processes such as cell signalling, immune inflammatory responses, or apoptosis, all of them key mechanisms involved in atherothrombosis. Moreover, since TRX and PRDX are present in the pathological vascular wall and can be secreted under prooxidative conditions to the circulation, several studies have addressed their role as diagnostic, prognostic, and therapeutic biomarkers of cardiovascular diseases (CVDs). PMID:22792412

  6. The Penicillium digitatum protein O-mannosyltransferase Pmt2 is required for cell wall integrity, conidiogenesis, virulence and sensitivity to the antifungal peptide PAF26.

    PubMed

    Harries, Eleonora; Gandía, Mónica; Carmona, Lourdes; Marcos, Jose F

    2015-09-01

    The activity of protein O-mannosyltransferases (Pmts) affects the morphogenesis and virulence of fungal pathogens. Recently, PMT genes have been shown to determine the sensitivity of Saccharomyces cerevisiae to the antifungal peptide PAF26. This study reports the identification and characterization of the three Pdpmt genes in the citrus post-harvest pathogen Penicillium digitatum. The Pdpmt genes are expressed during fungal growth and fruit infection, with the highest induction for Pdpmt2. Pdpmt2 complemented the growth defect of the S. cerevisiae Δpmt2 strain. The Pdpmt2 gene mutation in P. digitatum caused pleiotropic effects, including a reduction in fungal growth and virulence, whereas its constitutive expression had no phenotypic effect. The Pdpmt2 null mutants also showed a distinctive colourless phenotype with a strong reduction in the number of conidia, which was associated with severe alterations in the development of conidiophores. Additional effects of the Pdpmt2 mutation were hyphal morphological alterations, increased sensitivity to cell wall-interfering compounds and a blockage of invasive growth. In contrast, the Pdpmt2 mutation increased tolerance to oxidative stress and to the antifungal activity of PAF26. These data confirm the role of protein O-glycosylation in the PAF26-mediated antifungal mechanism present in distantly related fungal species. Important to future crop protection strategies, this study demonstrates that a mutation rendering fungi more resistant to an antifungal peptide results in severe deleterious effects on fungal growth and virulence. PMID:25640475

  7. Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

    PubMed Central

    Besingi, Richard N.; Clark, Patricia L.

    2016-01-01

    Membrane proteins play crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but is rapidly digested when cells are lysed prior to evaluation. Reporter proteins that are resistant to proteases (e.g. maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g. SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h. PMID:26584447

  8. Protein tyrosine nitration in the cell cycle

    SciTech Connect

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-09-23

    Highlights: {yields} Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. {yields} Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. {yields} 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.

  9. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  10. Integrated bioprocessing for plant cell cultures.

    PubMed

    Choi, J W; Cho, G H; Byun, S Y; Kim, D I

    2001-01-01

    Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design. Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture. Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity. In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents. In situ adsorption using polymeric resins is another possibility. Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity. In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed. PMID:11729756

  11. Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device.

    PubMed

    Yu, Ting; Guo, Zhe; Fan, Hui; Song, Jing; Liu, Yuanbin; Gao, Zhancheng; Wang, Qi

    2016-05-01

    The tumor microenvironment is comprised of cancer cells and various stromal cells and their respective cellular components. Cancer-associated fibroblasts (CAFs), a major part of the stromal cells, are a key determinant in tumor progression, while glucose-regulated protein (GRP)78 is overexpressed in many human cancers and is involved in tumor invasion and metastasis. This study developed a microfluidic-based three dimension (3D) co-culture device to mimic an in vitro tumor microenvironment in order to investigate tumor cell invasion in real-time. This bionic chip provided significant information regarding the role of GRP78, which may be stimulated by CAFs, to promote non-small cell lung cancer cell invasion in vitro. The data showed that CAF induced migration of NSCLC A549 and SPCA-1 cells in this three-dimensional invasion microdevice, which is confirmed by using the traditional Transwell system. Furthermore, CAF induced GRP78 expression in A549 and SPCA-1 cells to facilitate NSCLC cell migration and invasion, whereas knockdown of GRP78 expression blocked A549 and SPCA-1 cell migration and invasion capacity. In conclusion, these data indicated that CAFs might promote NSCLC cell invasion by up-regulation of GRP78 expression and this bionic chip microdevice is a robust platform to assess the interaction of cancer and stromal cells in tumor environment study. PMID:27016417

  12. 3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy.

    PubMed

    Pan, Bin-Bin; Yang, Feng; Ye, Yansheng; Wu, Qiong; Li, Conggang; Huber, Thomas; Su, Xun-Cheng

    2016-08-11

    Determining the three-dimensional structure of a protein in living cells remains particularly challenging. We demonstrated that the integration of site-specific tagging proteins and GPS-Rosetta calculations provides a fast and effective way of determining the structures of proteins in living cells, and in principle the interactions and dynamics of protein-ligand complexes. PMID:27470136

  13. p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses.

    PubMed

    Hünten, Sabine; Kaller, Markus; Drepper, Friedel; Oeljeklaus, Silke; Bonfert, Thomas; Erhard, Florian; Dueck, Anne; Eichner, Norbert; Friedel, Caroline C; Meister, Gunter; Zimmer, Ralf; Warscheid, Bettina; Hermeking, Heiko

    2015-10-01

    We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the

  14. Protein folding. Translational tuning optimizes nascent protein folding in cells.

    PubMed

    Kim, Soo Jung; Yoon, Jae Seok; Shishido, Hideki; Yang, Zhongying; Rooney, LeeAnn A; Barral, Jose M; Skach, William R

    2015-04-24

    In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/β-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating β-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis. PMID:25908822

  15. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  16. A Mass Spectrometric-Derived Cell Surface Protein Atlas

    PubMed Central

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P.; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L.; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E.; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R.; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  17. Integrated bioethanol and protein production from brown seaweed Laminaria digitata.

    PubMed

    Hou, Xiaoru; Hansen, Jonas Høeg; Bjerre, Anne-Belinda

    2015-12-01

    A wild-growing glucose-rich (i.e. 56.7% glucose content) brown seaweed species Laminaria digitata, collected from the North Coast of Denmark in August 2012, was used as the feedstock for an integrated bioethanol and protein production. Glutamic acid and aspartic acid are the two most abundant amino acids in the algal protein, both with proportional content of 10% in crude protein. Only minor pretreatment of milling was used on the biomass to facilitate the subsequent enzymatic hydrolysis and fermentation. The Separate Hydrolysis and Fermentation (SHF) resulted in obviously higher ethanol yield than the Simultaneous Saccharification and Fermentation (SSF). High conversion rate at maximum of 84.1% glucose recovery by enzymatic hydrolysis and overall ethanol yield at maximum of 77.7% theoretical were achieved. Protein content in the solid residues after fermentation was enriched by 2.7 fold, with similar distributions of amino acids, due to the hydrolysis of polymers in the seaweed cell wall matrix. PMID:26342344

  18. Integrated imaging instrument for self-calibrated fluorescence protein microarrays

    NASA Astrophysics Data System (ADS)

    Reddington, A. P.; Monroe, M. R.; Ünlü, M. S.

    2013-10-01

    Protein microarrays, or multiplexed and high-throughput assays, monitor multiple protein binding events to facilitate the understanding of disease progression and cell physiology. Fluorescence imaging is a popular method to detect proteins captured by immobilized probes with high sensitivity and specificity. Reliability of fluorescence assays depends on achieving minimal inter- and intra-assay probe immobilization variation, an ongoing challenge for protein microarrays. Therefore, it is desirable to establish a label-free method to quantify the probe density prior to target incubation to calibrate the fluorescence readout. Previously, a silicon oxide on silicon chip design was introduced to enhance the fluorescence signal and enable interferometric imaging to self-calibrate the signal with the immobilized probe density. In this paper, an integrated interferometric reflectance imaging sensor and wide-field fluorescence instrument is introduced for sensitive and calibrated microarray measurements. This platform is able to analyze a 2.5 mm × 3.4 mm area, or 200 spots (100 μm diameter with 200 μm pitch), in a single field-of-view.

  19. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

  20. Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry

    SciTech Connect

    Blonder, Josip; Goshe, Michael B.; Moore, Ronald J.; Pasa-Tolic, Liljiana; Masselon, Christophe D.; Lipton, Mary S.; Smith, Richard D.

    2002-04-01

    Currently, most proteomic studies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents, chaotropes, or reducing reagents that often interfere with electrospray ionization (ESI). To increase the identification of integral membrane proteins by LC-ESI-MS/MS, a sample preparation method combining carbonate extraction and surfactant-free organics solvent-assisted solubilization and proteolysis was developed and used to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic based on their positive grand average of hydropathicity values that covers 15% of the theoretical hydrophobic proteome. Using the PSORT algorithm, 268 identified proteins were recognized as integral membrane proteins covering 21% and 43% of the predicted integral cytoplasmic and outer membrane proteins, respectively. Of the integral cytoplasmic membrane proteins containing four or more predicted transmembrane domains (TMDs), 65% were identified by detecting at least one peptide spanning a TMD using LC-MS/MS. The extensive identification of highly hydrophobic proteins containing multiple TMDs confirms the efficacy of the described sample preparation protocol to isolate and solubilize integral membrane proteins and validates the method for large-scale analysis of bacterial membrane subproteomes using LC-ESI-MS/MS.

  1. Alterations in cell adhesion proteins and cardiomyopathy

    PubMed Central

    Li, Jifen

    2014-01-01

    Cell adhesive junction is specialized intercellular structure composed of cell adhesion proteins. They are essential to connect adjacent heart muscle cell and make heart contraction effectively and properly. Clinical and genetic studies have revealed close relationship between cell adhesive proteins and the occurrence of various cardiomyopathies. Here we will review recent development on the disease phenotype, potential cellular and molecular mechanism related to cell adhesion molecules, with particular disease pathogenesis learned from genetic manipulated murine models. PMID:24944760

  2. Fundamentals of fuel cell system integration

    NASA Astrophysics Data System (ADS)

    Krumpelt, Michael; Kumar, Romesh; Myles, Kevin M.

    1994-04-01

    Fuel cells are theoretically very efficient energy conversion devices that have the potential of becoming a commercial product for numerous uses in the civilian economy. We have analyzed several fuel cell system designs with regard to thermal and chemical integration of the fuel cell stack into the rest of the system. Thermal integration permits the use of the stack waste heat for the endothermic steps of fuel reforming. Chemical integration provides the steam needed for fuel reforming from the water produced by the electrochemical cell reaction. High-temperature fuel cells, such as the molten carbonate and the solid oxide fuel cells, permit this system integration in a relatively simple manner. Lower temperature fuel cells, such as the polymer electrolyte and phosphoric acid systems, require added system complexity to achieve such integration. The system economics are affected by capital and fuel costs and technical parameters, such as electrochemical fuel utilization, current density, and system complexity. At today's low fuel prices and the high fuel cell costs (in part, because of the low rates of production of the early prototypes), fuel cell systems are not cost competitive with conventional power generation. With the manufacture and sale of larger numbers of fuel cell systems, the total costs will decrease from the current several thousand dollars per kW, to perhaps less than $100 per kW as production volumes approa ch a million units per year.

  3. Fuel cell integrated with steam reformer

    DOEpatents

    Beshty, Bahjat S.; Whelan, James A.

    1987-01-01

    A H.sub.2 -air fuel cell integrated with a steam reformer is disclosed wherein a superheated water/methanol mixture is fed to a catalytic reformer to provide a continuous supply of hydrogen to the fuel cell, the gases exhausted from the anode of the fuel cell providing the thermal energy, via combustion, for superheating the water/methanol mixture.

  4. Large-scale protein-protein interactions detection by integrating big biosensing data with computational model.

    PubMed

    You, Zhu-Hong; Li, Shuai; Gao, Xin; Luo, Xin; Ji, Zhen

    2014-01-01

    Protein-protein interactions are the basis of biological functions, and studying these interactions on a molecular level is of crucial importance for understanding the functionality of a living cell. During the past decade, biosensors have emerged as an important tool for the high-throughput identification of proteins and their interactions. However, the high-throughput experimental methods for identifying PPIs are both time-consuming and expensive. On the other hand, high-throughput PPI data are often associated with high false-positive and high false-negative rates. Targeting at these problems, we propose a method for PPI detection by integrating biosensor-based PPI data with a novel computational model. This method was developed based on the algorithm of extreme learning machine combined with a novel representation of protein sequence descriptor. When performed on the large-scale human protein interaction dataset, the proposed method achieved 84.8% prediction accuracy with 84.08% sensitivity at the specificity of 85.53%. We conducted more extensive experiments to compare the proposed method with the state-of-the-art techniques, support vector machine. The achieved results demonstrate that our approach is very promising for detecting new PPIs, and it can be a helpful supplement for biosensor-based PPI data detection. PMID:25215285

  5. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  6. Integrated Protein-Crystal-Growing Apparatus

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.; Pusey, Marc L.

    1991-01-01

    Proposed apparatus for research on growth of protein crystals dispenses drops of protein and precipitating solutions, provides controlled environment for crystalization, and stores crystals. Intended for use in microgravity of outer space, concept of apparatus also useful in design of self-contained terrestrial experiments for remote and/or automatic execution.

  7. 14-3-3 Proteins in Guard Cell Signaling

    PubMed Central

    Cotelle, Valérie; Leonhardt, Nathalie

    2016-01-01

    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. PMID:26858725

  8. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

  9. The BET family of proteins targets Moloney Murine Leukemia Virus integration near transcription start sites

    PubMed Central

    De Rijck, Jan; de Kogel, Christine; Demeulemeester, Jonas; Vets, Sofie; Ashkar, Sara El; Malani, Nirav; Bushman, Frederic D; Landuyt, Bart; Husson, Steven J.; Busschots, Katrien; Gijsbers, Rik; Debyser, Zeger

    2014-01-01

    Summary A hallmark of retroviral replication is integration of the viral genome in the host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to Murine Leukemia Virus (MLV)-derived vector integration, hamper their application. Here we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3 and BRD4) and MLV integrase specifically interact and co-localize within the nucleus of the cell. Inhibition of the BET proteins chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration away from TSS and into the body of actively transcribed genes, conform to the Human Immunodeficiency Virus (HIV) integration pattern. Together these data validate BET proteins as MLV integration targeting factors. PMID:24183673

  10. Differential protein network analysis of the immune cell lineage.

    PubMed

    Clancy, Trevor; Hovig, Eivind

    2014-01-01

    Recently, the Immunological Genome Project (ImmGen) completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks. PMID:25309909

  11. Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy

    PubMed Central

    Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J.; Auchus, Richard J.; DeLisa, Matthew P.

    2015-01-01

    Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic ‘shield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941

  12. Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy.

    PubMed

    Mizrachi, Dario; Chen, Yujie; Liu, Jiayan; Peng, Hwei-Ming; Ke, Ailong; Pollack, Lois; Turner, Raymond J; Auchus, Richard J; DeLisa, Matthew P

    2015-01-01

    Integral membrane proteins (IMPs) play crucial roles in all cells and represent attractive pharmacological targets. However, functional and structural studies of IMPs are hindered by their hydrophobic nature and the fact that they are generally unstable following extraction from their native membrane environment using detergents. Here we devise a general strategy for in vivo solubilization of IMPs in structurally relevant conformations without the need for detergents or mutations to the IMP itself, as an alternative to extraction and in vitro solubilization. This technique, called SIMPLEx (solubilization of IMPs with high levels of expression), allows the direct expression of soluble products in living cells by simply fusing an IMP target with truncated apolipoprotein A-I, which serves as an amphipathic proteic 'shield' that sequesters the IMP from water and promotes its solubilization. PMID:25851941

  13. TAK1 regulates Paneth cell integrity partly through blocking necroptosis

    PubMed Central

    Simmons, A N; Kajino-Sakamoto, R; Ninomiya-Tsuji, J

    2016-01-01

    Paneth cells reside at the base of crypts of the small intestine and secrete antimicrobial factors to control gut microbiota. Paneth cell loss is observed in the chronically inflamed intestine, which is often associated with increased reactive oxygen species (ROS). However, the relationship between Paneth cell loss and ROS is not yet clear. Intestinal epithelial-specific deletion of a protein kinase Tak1 depletes Paneth cells and highly upregulates ROS in the mouse model. We found that depletion of gut bacteria or myeloid differentiation factor 88 (Myd88), a mediator of bacteria-derived cell signaling, reduced ROS but did not block Paneth cell loss, suggesting that gut bacteria are the cause of ROS accumulation but bacteria-induced ROS are not the cause of Paneth cell loss. In contrast, deletion of the necroptotic cell death signaling intermediate, receptor-interacting protein kinase 3 (Ripk3), partially blocked Paneth cell loss. Thus, Tak1 deletion causes Paneth cell loss in part through necroptotic cell death. These results suggest that TAK1 participates in intestinal integrity through separately modulating bacteria-derived ROS and RIPK3-dependent Paneth cell loss. PMID:27077812

  14. Obtaining a protein concentrate from integral defatted sunflower flour.

    PubMed

    Ordóñez, C; Asenjo, M G; Benitez, C; González, J L

    2001-06-01

    Proteins which are found in integral defatted sunflower flour (27% of protein in dry weight) allow us to produce a protein concentrate by means of extraction of proteins with a basic pH solution, followed by their precipitation with an acid pH solution. Once the suitable conditions for pH and temperature were fixed in order to carry out these processes, a solid proteic concentrate (71% of protein in dry weight) was obtained which was rich in glutamic and aspartic acids, with a liquid supernatant very rich in phosphorus and potassium, which might be used as an agricultural fertilizer. PMID:11333039

  15. Protein-based integrated optical switching and modulation

    NASA Astrophysics Data System (ADS)

    Ormos, Pál; Fábián, László; Oroszi, László; Wolff, Elmar K.; Ramsden, Jeremy J.; Dér, András

    2002-05-01

    The static and dynamic response of optical waveguides coated with a thin protein film of bacteriorhodopsin was investigated. The size and kinetics of the light-induced refractive index changes of the adlayer were determined under different conditions of illumination. The results demonstrate the applicability of this protein as an active, programmable nonlinear optical material in all-optical integrated circuits.

  16. NetworkAnalyst--integrative approaches for protein-protein interaction network analysis and visual exploration.

    PubMed

    Xia, Jianguo; Benner, Maia J; Hancock, Robert E W

    2014-07-01

    Biological network analysis is a powerful approach to gain systems-level understanding of patterns of gene expression in different cell types, disease states and other biological/experimental conditions. Three consecutive steps are required--identification of genes or proteins of interest, network construction and network analysis and visualization. To date, researchers have to learn to use a combination of several tools to accomplish this task. In addition, interactive visualization of large networks has been primarily restricted to locally installed programs. To address these challenges, we have developed NetworkAnalyst, taking advantage of state-of-the-art web technologies, to enable high performance network analysis with rich user experience. NetworkAnalyst integrates all three steps and presents the results via a powerful online network visualization framework. Users can upload gene or protein lists, single or multiple gene expression datasets to perform comprehensive gene annotation and differential expression analysis. Significant genes are mapped to our manually curated protein-protein interaction database to construct relevant networks. The results are presented through standard web browsers for network analysis and interactive exploration. NetworkAnalyst supports common functions for network topology and module analyses. Users can easily search, zoom and highlight nodes or modules, as well as perform functional enrichment analysis on these selections. The networks can be customized with different layouts, colors or node sizes, and exported as PNG, PDF or GraphML files. Comprehensive FAQs, tutorials and context-based tips and instructions are provided. NetworkAnalyst currently supports protein-protein interaction network analysis for human and mouse and is freely available at http://www.networkanalyst.ca. PMID:24861621

  17. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

  18. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    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).

  19. Development of Lentiviral Vectors for Targeted Integration and Protein Delivery.

    PubMed

    Schenkwein, Diana; Ylä-Herttuala, Seppo

    2016-01-01

    The method in this chapter describes the design of human immunodeficiency virus type 1 (HIV-1) integrase (IN)-fusion proteins which we have developed to transport different proteins into the nuclei of lentiviral vector (LV)-transduced cells. The IN-fusion protein cDNA is incorporated into the LV packaging plasmid, which leads to its incorporation into vector particles as part of a large Gag-Pol polyprotein. This specific feature of protein packaging enables also the incorporation of cytotoxic and proapoptotic proteins, such as frequently cutting endonucleases and P53. The vectors can hence be used for various protein transduction needs. An outline of the necessary methods is also given to study the functionality of a chosen IN-fusion protein in a cell culture assay. PMID:27317182

  20. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    PubMed

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  1. Integrative analysis of human protein, function and disease networks

    PubMed Central

    Liu, Wei; Wu, Aiping; Pellegrini, Matteo; Wang, Xiaofan

    2015-01-01

    Protein-protein interaction (PPI) networks serve as a powerful tool for unraveling protein functions, disease-gene and disease-disease associations. However, a direct strategy for integrating protein interaction, protein function and diseases is still absent. Moreover, the interrelated relationships among these three levels are poorly understood. Here we present a novel systematic method to integrate protein interaction, function, and disease networks. We first identified topological modules in human protein interaction data using the network topological algorithm (NeTA) we previously developed. The resulting modules were then associated with functional terms using Gene Ontology to obtain functional modules. Finally, disease modules were constructed by associating the modules with OMIM and GWAS. We found that most topological modules have cohesive structure, significant pathway annotations and good modularity. Most functional modules (70.6%) fully cover corresponding topological modules, and most disease modules (88.5%) are fully covered by the corresponding functional modules. Furthermore, we identified several protein modules of interest that we describe in detail, which demonstrate the power of our integrative approach. This approach allows us to link genes, and pathways with their corresponding disorders, which may ultimately help us to improve the prevention, diagnosis and treatment of disease. PMID:26399914

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

    PubMed Central

    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

  3. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. PMID:24136528

  4. Cell and organ printing 1: protein and cell printers.

    PubMed

    Wilson, W Cris; Boland, Thomas

    2003-06-01

    We have developed several devices for positioning organic molecules, molecular aggregates, cells, and single-cell organisms onto solid supports. These printers can create stable, functional protein arrays using an inexpensive technology. The cell printer allows us to create cell libraries as well as cellular assemblies that mimic their respective position in organs. The printers are derived from commercially available ink-jet printers that are modified to dispense protein or cell solutions instead of ink. We describe here the modifications to the print heads, and the printer hardware and software that enabled us to adapt the ink-jet printers for the manufacture of cell and protein arrays. The printers have the advantage of being fully automated and computer controlled, and allow for the high-throughput manufacture of protein and cell arrays. PMID:12740942

  5. STRING v9.1: protein-protein interaction networks, with increased coverage and integration

    PubMed Central

    Franceschini, Andrea; Szklarczyk, Damian; Frankild, Sune; Kuhn, Michael; Simonovic, Milan; Roth, Alexander; Lin, Jianyi; Minguez, Pablo; Bork, Peer; von Mering, Christian; Jensen, Lars J.

    2013-01-01

    Complete knowledge of all direct and indirect interactions between proteins in a given cell would represent an important milestone towards a comprehensive description of cellular mechanisms and functions. Although this goal is still elusive, considerable progress has been made—particularly for certain model organisms and functional systems. Currently, protein interactions and associations are annotated at various levels of detail in online resources, ranging from raw data repositories to highly formalized pathway databases. For many applications, a global view of all the available interaction data is desirable, including lower-quality data and/or computational predictions. The STRING database (http://string-db.org/) aims to provide such a global perspective for as many organisms as feasible. Known and predicted associations are scored and integrated, resulting in comprehensive protein networks covering >1100 organisms. Here, we describe the update to version 9.1 of STRING, introducing several improvements: (i) we extend the automated mining of scientific texts for interaction information, to now also include full-text articles; (ii) we entirely re-designed the algorithm for transferring interactions from one model organism to the other; and (iii) we provide users with statistical information on any functional enrichment observed in their networks. PMID:23203871

  6. MALDI Tissue Profiling of Integral Membrane Proteins from Ocular Tissues

    PubMed Central

    Thibault, Danielle B.; Gillam, Christopher J.; Grey, Angus C.; Han, Jun; Schey, Kevin L.

    2008-01-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this communication, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed aged related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods. PMID:18396059

  7. Integrated process for high conversion and high yield protein PEGylation.

    PubMed

    Pfister, David; Morbidelli, Massimo

    2016-08-01

    Over the past decades, PEGylation has become a powerful technique to increase the in vivo circulation half-life of therapeutic proteins while maintaining their activity. The development of new therapeutic proteins is likely to require further improvement of the PEGylation methods to reach even better selectivity and yield for reduced costs. The intensification of the PEGylation process was investigated through the integration of a chromatographic step in order to increase yield and conversion for the production of mono-PEGylated protein. Lysozyme was used as a model protein to demonstrate the feasibility of such approach. In the integrated reaction/separation process, chromatography was used as fractionation technique in order to isolate and recycle the unreacted protein from the PEGylated products. This allows operating the reactor with short reaction times so as to minimize the production of multi-PEGylated proteins (i.e., conjugated to more than one polymer). That is, the reaction is stopped before the desired product (i.e., the mono-PEGylated protein) can further react, thus leading to limited conversion but high yield. The recycling of the unreacted protein was then considered to drive the protein overall conversion to completion. This approach has great potential to improve processes whose yield is limited by the further reaction of the product leading to undesirable by-products. Biotechnol. Bioeng. 2016;113: 1711-1718. © 2016 Wiley Periodicals, Inc. PMID:26757029

  8. A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane

    SciTech Connect

    Bruce, Lesley J.; Beckmann, Roland; Ribeiro, M. Leticia; Peters, Luanne L.; Chasis, Joel A.; Delaunay, Jean; Mohandas, Narla; Anstee, David J.; Tanner, Michael J.A.

    2003-06-18

    We have studied the membrane proteins of band 3 anion exchanger (AE1)-deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3 RBCs differed from the human band 3-deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3-deficient RBCs. Proteins of the glycophorin complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural Macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO2/O2 gas exchange unit (metabolon) in the erythrocyte.

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

    SciTech Connect

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

    1988-02-01

    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 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 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 /sup 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 (/sup 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.

  10. Integrating Sphere Alkali-Metal Vapor Cells

    NASA Astrophysics Data System (ADS)

    McGuyer, Bart; Ben-Kish, Amit; Jau, Yuan-Yu; Happer, William

    2010-03-01

    An integrating sphere is an optical multi-pass cavity that uses diffuse reflection to increase the optical path length. Typically applied in photometry and radiometry, integrating spheres have previously been used to detect trace gases and to cool and trap alkali-metal atoms. Here, we investigate the potential for integrating spheres to enhance optical absorption in optically thin alkali-metal vapor cells. In particular, we consider the importance of dielectric effects due to a glass container for the alkali-metal vapor. Potential applications include miniature atomic clocks and magnetometers, where multi-passing could reduce the operating temperature and power consumption.

  11. Cell-free protein synthesis in microfluidic array devices.

    PubMed

    Mei, Qian; Fredrickson, Carl K; Simon, Andrew; Khnouf, Ruba; Fan, Z Hugh

    2007-01-01

    We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield. PMID:17924644

  12. Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen-activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae.

    PubMed

    Yin, Ziyi; Tang, Wei; Wang, Jingzhen; Liu, Xinyu; Yang, Lina; Gao, Chuyun; Zhang, Jinlong; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2016-06-01

    In the rice blast fungus Magnaporthe oryzae, the high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen-activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1-MoMkk1-MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)-induced UPR pathway in M. oryzae. PMID:27193947

  13. INTEGRATING COMPUTATIONAL PROTEIN FUNCTION PREDICTION INTO DRUG DISCOVERY INITIATIVES

    PubMed Central

    Grant, Marianne A.

    2014-01-01

    Pharmaceutical researchers must evaluate vast numbers of protein sequences and formulate innovative strategies for identifying valid targets and discovering leads against them as a way of accelerating drug discovery. The ever increasing number and diversity of novel protein sequences identified by genomic sequencing projects and the success of worldwide structural genomics initiatives have spurred great interest and impetus in the development of methods for accurate, computationally empowered protein function prediction and active site identification. Previously, in the absence of direct experimental evidence, homology-based protein function annotation remained the gold-standard for in silico analysis and prediction of protein function. However, with the continued exponential expansion of sequence databases, this approach is not always applicable, as fewer query protein sequences demonstrate significant homology to protein gene products of known function. As a result, several non-homology based methods for protein function prediction that are based on sequence features, structure, evolution, biochemical and genetic knowledge have emerged. Herein, we review current bioinformatic programs and approaches for protein function prediction/annotation and discuss their integration into drug discovery initiatives. The development of such methods to annotate protein functional sites and their application to large protein functional families is crucial to successfully utilizing the vast amounts of genomic sequence information available to drug discovery and development processes. PMID:25530654

  14. Targeting Cell Survival Proteins for Cancer Cell Death

    PubMed Central

    Pandey, Manoj K.; Prasad, Sahdeo; Tyagi, Amit Kumar; Deb, Lokesh; Huang, Jiamin; Karelia, Deepkamal N.; Amin, Shantu G.; Aggarwal, Bharat B.

    2016-01-01

    Escaping from cell death is one of the adaptations that enable cancer cells to stave off anticancer therapies. The key players in avoiding apoptosis are collectively known as survival proteins. Survival proteins comprise the Bcl-2, inhibitor of apoptosis (IAP), and heat shock protein (HSP) families. The aberrant expression of these proteins is associated with a range of biological activities that promote cancer cell survival, proliferation, and resistance to therapy. Several therapeutic strategies that target survival proteins are based on mimicking BH3 domains or the IAP-binding motif or competing with ATP for the Hsp90 ATP-binding pocket. Alternative strategies, including use of nutraceuticals, transcriptional repression, and antisense oligonucleotides, provide options to target survival proteins. This review focuses on the role of survival proteins in chemoresistance and current therapeutic strategies in preclinical or clinical trials that target survival protein signaling pathways. Recent approaches to target survival proteins-including nutraceuticals, small-molecule inhibitors, peptides, and Bcl-2-specific mimetic are explored. Therapeutic inventions targeting survival proteins are promising strategies to inhibit cancer cell survival and chemoresistance. However, complete eradication of resistance is a distant dream. For a successful clinical outcome, pretreatment with novel survival protein inhibitors alone or in combination with conventional therapies holds great promise. PMID:26927133

  15. Targeting Cell Survival Proteins for Cancer Cell Death.

    PubMed

    Pandey, Manoj K; Prasad, Sahdeo; Tyagi, Amit Kumar; Deb, Lokesh; Huang, Jiamin; Karelia, Deepkamal N; Amin, Shantu G; Aggarwal, Bharat B

    2016-01-01

    Escaping from cell death is one of the adaptations that enable cancer cells to stave off anticancer therapies. The key players in avoiding apoptosis are collectively known as survival proteins. Survival proteins comprise the Bcl-2, inhibitor of apoptosis (IAP), and heat shock protein (HSP) families. The aberrant expression of these proteins is associated with a range of biological activities that promote cancer cell survival, proliferation, and resistance to therapy. Several therapeutic strategies that target survival proteins are based on mimicking BH3 domains or the IAP-binding motif or competing with ATP for the Hsp90 ATP-binding pocket. Alternative strategies, including use of nutraceuticals, transcriptional repression, and antisense oligonucleotides, provide options to target survival proteins. This review focuses on the role of survival proteins in chemoresistance and current therapeutic strategies in preclinical or clinical trials that target survival protein signaling pathways. Recent approaches to target survival proteins-including nutraceuticals, small-molecule inhibitors, peptides, and Bcl-2-specific mimetic are explored. Therapeutic inventions targeting survival proteins are promising strategies to inhibit cancer cell survival and chemoresistance. However, complete eradication of resistance is a distant dream. For a successful clinical outcome, pretreatment with novel survival protein inhibitors alone or in combination with conventional therapies holds great promise. PMID:26927133

  16. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

    SciTech Connect

    Goshe, Michael B.; Blonder, Josip; Smith, Richard D.

    2003-03-01

    The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

  17. Integrated regenerative fuel cell experimental evaluation

    NASA Technical Reports Server (NTRS)

    Martin, Ronald E.

    1990-01-01

    An experimental test program was conducted to investigate the performance characteristics of an integrated regenerative fuel cell (IRFC) concept. The IRFC consists of a separate fuel cell unit and electrolysis cell unit in the same structure, with internal storage of fuel cell product water and external storage of electrolysis cell produced hydrogen and oxygen. The fuel cell unit incorporates an enhanced Orbiter-type cell capable of improved performance at reduced weight. The electrolysis cell features a NiCo2O4 catalyst oxygen evolution eletrode with a porous Teflon cover to retard electrolyte loss. Six complete IRFC assemblies were assembled and performance tested at an operating temperature of 200 F (93.3 C) and reactant pressures up to 170 psia (117.2 n/cu cm) on IRFC No. 4. Anomalous pressure charge/discharge characteristics were encountered during performance evaluation. A reversible fuel cell incorporating a proprietary bi-functional oxygen electrode operated satisfactory at 200 F (93.3 C) at reactant pressures up to 50 psia (41.4 n/cu cm) as a regenerative fuel cell for one cycle, before developing an electrical short in the fuel cell mode. Electrolysis cell 300-hour endurance tests demonstrated the electrolyte retention capability of the electrode Teflon cover and the performance stability of the bi-functional oxygen electrode at high potential.

  18. Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.

    PubMed

    Lee, Jin-Gu; Takahama, Shokichi; Zhang, Guofeng; Tomarev, Stanislav I; Ye, Yihong

    2016-07-01

    To safeguard proteomic integrity, cells rely on the proteasome to degrade aberrant polypeptides, but it is unclear how cells remove defective proteins that have escaped degradation owing to proteasome insufficiency or dysfunction. Here we report a pathway termed misfolding-associated protein secretion, which uses the endoplasmic reticulum (ER)-associated deubiquitylase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitylation. Deubiquitylated cargos are encapsulated into ER-associated late endosomes and secreted to the cell exterior. USP19-deficient cells cannot efficiently secrete unwanted proteins, and grow more slowly than wild-type cells following exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway that, unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process. PMID:27295555

  19. Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

    PubMed

    Focke, Paul J; Hein, Christopher; Hoffmann, Beate; Matulef, Kimberly; Bernhard, Frank; Dötsch, Volker; Valiyaveetil, Francis I

    2016-08-01

    Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence. PMID:27384110

  20. Comparative genomic analysis of integral membrane transport proteins in ciliates.

    PubMed

    Kumar, Ujjwal; Saier, Milton H

    2015-01-01

    Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism. PMID:25099884

  1. Cell-free protein expression based on extracts from CHO cells.

    PubMed

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins. PMID:24018795

  2. Genome integrity, stem cells and hyaluronan

    PubMed Central

    Darzynkiewicz, Zbigniew; Balazs, Endre A.

    2012-01-01

    Faithful preservation of genome integrity is the critical mission of stem cells as well as of germ cells. Reviewed are the following mechanisms involved in protecting DNA in these cells: (a) The efflux machinery that can pump out variety of genotoxins in ATP-dependent manner; (b) the mechanisms maintaining minimal metabolic activity which reduces generation of reactive oxidants, by-products of aerobic respiration; (c) the role of hypoxic niche of stem cells providing a gradient of variable oxygen tension; (d) (e) the presence of hyaluronan (HA) and HA receptors on stem cells and in the niche; (f) the role of HA in protecting DNA from oxidative damage; (g) the specific function of HA in protecting DNA in stem cells; (h) the interactions of HA with sperm cells and oocytes that also may shield their DNA from oxidative damage, and (e) mechanisms by which HA exerts the anti-oxidant activity. While HA has multitude of functions its anti-oxidant capabilities are often overlooked but may be of significance in preservation of integrity of stem and germ cells genome. PMID:22383371

  3. A protein interaction map for cell polarity development.

    PubMed

    Drees, B L; Sundin, B; Brazeau, E; Caviston, J P; Chen, G C; Guo, W; Kozminski, K G; Lau, M W; Moskow, J J; Tong, A; Schenkman, L R; McKenzie, A; Brennwald, P; Longtine, M; Bi, E; Chan, C; Novick, P; Boone, C; Pringle, J R; Davis, T N; Fields, S; Drubin, D G

    2001-08-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  4. Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.

    PubMed

    Bird, Jonathan E; Daudet, Nicolas; Warchol, Mark E; Gale, Jonathan E

    2010-09-15

    Epithelial homeostasis is essential for sensory transduction in the auditory and vestibular organs of the inner ear, but how it is maintained during trauma is poorly understood. To examine potential repair mechanisms, we expressed β-actin-enhanced green fluorescent protein (EGFP) in the chick inner ear and used live-cell imaging to study how sensory epithelia responded during aminoglycoside-induced hair cell trauma. We found that glial-like supporting cells used two independent mechanisms to rapidly eliminate dying hair cells. Supporting cells assembled an actin cable at the luminal surface that extended around the pericuticular junction and constricted to excise the stereocilia bundle and cuticular plate from the hair cell soma. Hair bundle excision could occur within 3 min of actin-cable formation. After bundle excision, typically with a delay of up to 2-3 h, supporting cells engulfed and phagocytosed the remaining bundle-less hair cell. Dual-channel recordings with β-actin-EGFP and vital dyes revealed phagocytosis was concurrent with loss of hair cell integrity. We conclude that supporting cells repaired the epithelial barrier before hair cell plasmalemmal integrity was lost and that supporting cell activity was closely linked to hair cell death. Treatment with the Rho-kinase inhibitor Y-27632 did not prevent bundle excision but prolonged phagocytic engulfment and resulted in hair cell corpses accumulating within the epithelium. Our data show that supporting cells not only maintain epithelial integrity during trauma but suggest they may also be an integral part of the hair cell death process itself. PMID:20844149

  5. Fragmentation of Integral Membrane Proteins in the Gas Phase

    PubMed Central

    2015-01-01

    Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement. PMID:24689519

  6. Technology Advancement for Integrative Stem Cell Analyses

    PubMed Central

    Jeong, Yoon

    2014-01-01

    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. PMID:24874188

  7. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    PubMed

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  8. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    PubMed

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  9. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  10. Origins of Protein Functions in Cells

    NASA Technical Reports Server (NTRS)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

  11. Cell-to-cell propagation of infectious cytosolic protein aggregates

    PubMed Central

    Hofmann, Julia P.; Denner, Philip; Nussbaum-Krammer, Carmen; Kuhn, Peer-Hendrik; Suhre, Michael H.; Scheibel, Thomas; Lichtenthaler, Stefan F.; Schätzl, Hermann M.; Bano, Daniele; Vorberg, Ina M.

    2013-01-01

    Prions are self-templating protein conformers that replicate by recruitment and conversion of homotypic proteins into growing protein aggregates. Originally identified as causative agents of transmissible spongiform encephalopathies, increasing evidence now suggests that prion-like phenomena are more common in nature than previously anticipated. In contrast to fungal prions that replicate in the cytoplasm, propagation of mammalian prions derived from the precursor protein PrP is confined to the cell membrane or endocytic vesicles. Here we demonstrate that cytosolic protein aggregates can also behave as infectious entities in mammalian cells. When expressed in the mammalian cytosol, protein aggregates derived from the prion domain NM of yeast translation termination factor Sup35 persistently propagate and invade neighboring cells, thereby inducing a self-perpetuating aggregation state of NM. Cell contact is required for efficient infection. Aggregates can also be induced in primary astrocytes, neurons, and organotypic cultures, demonstrating that this phenomenon is not specific to immortalized cells. Our data have important implications for understanding prion-like phenomena of protein aggregates associated with human diseases and for the growing number of amyloidogenic proteins discovered in mammals. PMID:23509289

  12. Protein diffusion in mammalian cell cytoplasm.

    PubMed

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS. PMID:21886771

  13. Reduced protein synthesis in schizophrenia patient-derived olfactory cells

    PubMed Central

    English, J A; Fan, Y; Föcking, M; Lopez, L M; Hryniewiecka, M; Wynne, K; Dicker, P; Matigian, N; Cagney, G; Mackay-Sim, A; Cotter, D R

    2015-01-01

    Human olfactory neurosphere-derived (ONS) cells have the potential to provide novel insights into the cellular pathology of schizophrenia. We used discovery-based proteomics and targeted functional analyses to reveal reductions in 17 ribosomal proteins, with an 18% decrease in the total ribosomal signal intensity in schizophrenia-patient-derived ONS cells. We quantified the rates of global protein synthesis in vitro and found a significant reduction in the rate of protein synthesis in schizophrenia patient-derived ONS cells compared with control-derived cells. Protein synthesis rates in fibroblast cell lines from the same patients did not differ, suggesting cell type-specific effects. Pathway analysis of dysregulated proteomic and transcriptomic data sets from these ONS cells converged to highlight perturbation of the eIF2α, eIF4 and mammalian target of rapamycin (mTOR) translational control pathways, and these pathways were also implicated in an independent induced pluripotent stem cell-derived neural stem model, and cohort, of schizophrenia patients. Analysis in schizophrenia genome-wide association data from the Psychiatric Genetics Consortium specifically implicated eIF2α regulatory kinase EIF2AK2, and confirmed the importance of the eIF2α, eIF4 and mTOR translational control pathways at the level of the genome. Thus, we integrated data from proteomic, transcriptomic, and functional assays from schizophrenia patient-derived ONS cells with genomics data to implicate dysregulated protein synthesis for the first time in schizophrenia. PMID:26485547

  14. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  15. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  16. Transduction of peptides and proteins into live cells by cell penetrating peptides.

    PubMed

    Mussbach, Franziska; Franke, Martin; Zoch, Ansgar; Schaefer, Buerk; Reissmann, Siegmund

    2011-12-01

    Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines. PMID:21826709

  17. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

    PubMed

    Brandt, M E; Riley, B S; Radolf, J D; Norgard, M V

    1990-04-01

    The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi. PMID:2318538

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

    PubMed

    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 i ntegrate 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. PMID:26800544

  19. Versatile protein tagging in cells with split fluorescent protein.

    PubMed

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A; Ishikawa, Hiroaki; Leonetti, Manuel D; Marshall, Wallace F; Weissman, Jonathan S; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  20. Versatile protein tagging in cells with split fluorescent protein

    PubMed Central

    Kamiyama, Daichi; Sekine, Sayaka; Barsi-Rhyne, Benjamin; Hu, Jeffrey; Chen, Baohui; Gilbert, Luke A.; Ishikawa, Hiroaki; Leonetti, Manuel D.; Marshall, Wallace F.; Weissman, Jonathan S.; Huang, Bo

    2016-01-01

    In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications. PMID:26988139

  1. Coal Integrated Gasification Fuel Cell System Study

    SciTech Connect

    Chellappa Balan; Debashis Dey; Sukru-Alper Eker; Max Peter; Pavel Sokolov; Greg Wotzak

    2004-01-31

    This study analyzes the performance and economics of power generation systems based on Solid Oxide Fuel Cell (SOFC) technology and fueled by gasified coal. System concepts that integrate a coal gasifier with a SOFC, a gas turbine, and a steam turbine were developed and analyzed for plant sizes in excess of 200 MW. Two alternative integration configurations were selected with projected system efficiency of over 53% on a HHV basis, or about 10 percentage points higher than that of the state-of-the-art Integrated Gasification Combined Cycle (IGCC) systems. The initial cost of both selected configurations was found to be comparable with the IGCC system costs at approximately $1700/kW. An absorption-based CO2 isolation scheme was developed, and its penalty on the system performance and cost was estimated to be less approximately 2.7% and $370/kW. Technology gaps and required engineering development efforts were identified and evaluated.

  2. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  3. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-06-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. PMID:3380805

  4. The adaptor protein Cindr regulates JNK activity to maintain epithelial sheet integrity.

    PubMed

    Yasin, Hannah W R; van Rensburg, Samuel H; Feiler, Christina E; Johnson, Ruth I

    2016-02-15

    Epithelia are essential barrier tissues that must be appropriately maintained for their correct function. To achieve this a plethora of protein interactions regulate epithelial cell number, structure and adhesion, and differentiation. Here we show that Cindr (the Drosophila Cin85 and Cd2ap ortholog) is required to maintain epithelial integrity. Reducing Cindr triggered cell delamination and movement. Most delaminating cells died. These behaviors were consistent with JNK activation previously associated with loss of epithelial integrity in response to ectopic oncogene activity. We confirmed a novel interaction between Cindr and Drosophila JNK (dJNK), which when perturbed caused inappropriate JNK signaling. Genetically reducing JNK signaling activity suppressed the effects of reducing Cindr. Furthermore, ectopic JNK signaling phenocopied loss of Cindr and was partially rescued by concomitant cindr over-expression. Thus, correct Cindr-dJNK stoichiometry is essential to maintain epithelial integrity and disturbing this balance may contribute to the pathogenesis of disease states, including cancer. PMID:26772997

  5. A protein interaction map for cell polarity development

    PubMed Central

    Drees, Becky L.; Sundin, Bryan; Brazeau, Elizabeth; Caviston, Juliane P.; Chen, Guang-Chao; Guo, Wei; Kozminski, Keith G.; Lau, Michelle W.; Moskow, John J.; Tong, Amy; Schenkman, Laura R.; McKenzie, Amos; Brennwald, Patrick; Longtine, Mark; Bi, Erfei; Chan, Clarence; Novick, Peter; Boone, Charles; Pringle, John R.; Davis, Trisha N.; Fields, Stanley; Drubin, David G.

    2001-01-01

    Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

  6. InterPro: the integrative protein signature database.

    PubMed

    Hunter, Sarah; Apweiler, Rolf; Attwood, Teresa K; Bairoch, Amos; Bateman, Alex; Binns, David; Bork, Peer; Das, Ujjwal; Daugherty, Louise; Duquenne, Lauranne; Finn, Robert D; Gough, Julian; Haft, Daniel; Hulo, Nicolas; Kahn, Daniel; Kelly, Elizabeth; Laugraud, Aurélie; Letunic, Ivica; Lonsdale, David; Lopez, Rodrigo; Madera, Martin; Maslen, John; McAnulla, Craig; McDowall, Jennifer; Mistry, Jaina; Mitchell, Alex; Mulder, Nicola; Natale, Darren; Orengo, Christine; Quinn, Antony F; Selengut, Jeremy D; Sigrist, Christian J A; Thimma, Manjula; Thomas, Paul D; Valentin, Franck; Wilson, Derek; Wu, Cathy H; Yeats, Corin

    2009-01-01

    The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or 'signatures' representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total approximately 58,000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein-protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/). PMID:18940856

  7. Coal Integrated Gasification Fuel Cell System Study

    SciTech Connect

    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.

  8. Cell loss in integrated microfluidic device.

    PubMed

    Zhu, Liang; Peh, Xue Li; Ji, Hong Miao; Teo, Cheng Yong; Feng, Han Hua; Liu, Wen-Tso

    2007-10-01

    Cell loss during sample transporting from macro-components to micro-components in integrated microfluidic devices can considerably deteriorate cell detection sensitivity. This intrinsic cell loss was studied and effectively minimized through (a) increasing the tubing diameter connecting the sample storage and the micro-device, (b) applying a hydrodynamic focusing approach for sample delivering to reduce cells contacting and adhesion on the walls of micro-channel and chip inlet; (c) optimizing the filter design with a zigzag arrangement of pillars (13 microm in chamber depth and 0.8 microm in gap) to prolong the effective filter length, and iv) the use of diamond shaped pillar instead of normally used rectangular shape to reduce the gap length between any two given pillar (i.e. pressure drop) at the filter region. Cell trapping and immunofluorescent detection of 12 Giardia lamblia and 12 Cryptosporidium parvum cells in 150 microl solution and 50 MCF-7 breast cancer cells in 150 microl solution was completed within 15 min with trapping efficiencies improved from 79+/-11%, 50.8+/-5.5% and 41.3+/-3.6% without hydrodynamic focusing, respectively, to 90.8+/-5.8%, 89.8+/-16.6% and 77.0+/-9.2% with hydrodynamic focusing. PMID:17541747

  9. ASIC proteins regulate smooth muscle cell migration.

    PubMed

    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. PMID:17936312

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

    PubMed

    Re, Angela; Workman, Christopher T; Waldron, Levi; Quattrone, Alessandro; Brunak, Søren

    2014-09-01

    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 programs. Functional analysis gathered insights in fate-specific candidates of interface functionalities. The non-transcriptionally regulated interface proteins were found to be highly regulated by post-translational ubiquitylation modification, which may synchronize the transition between cell proliferation and differentiation in ESCs. PMID:25173649

  11. Endocytosis of connexin protein in adrenal cells.

    PubMed

    Murray, Sandra A; Nickel, B M; Gay, V L

    2004-11-01

    The ability of adrenocorticotropin (ACTH) to affect gap junctions was examined in adrenal cells in vivo and in vitro. Treatment with ACTH increased the size and number of gap junction plaques on the cell membranes in hypophysectomized animals and in adrenal culture. Intracellular (cytoplasmic) annular gap junctions were observed in cells of the inner adrenal cortical zones and in adrenal cell cultures. To investigate the relationship of annular gap junctions to surface junctions, adrenal cells in culture were transfected with cDNA encoding a green fluorescent protein tagged connexin 43 construct (Cx43-GFP), and subsequently studied by time-lapse video microscopy, immunocytochemistry, and transmission electron microscopy (TEM). Internalization of part or all of a surface gap junction plaque resulted in annular gap junction formation. These studies support the hypothesis that cytoplasmic vesicles, initially described with TEM methods, can result from removal of gap junction plaques from the cell surface. It is suggested that hormones can play a regulatory role in cell-cell communication by influencing the availability of gap junction protein at the cell surface and that hormonally-sensitive processes might serve as a means of altering intercellular communication. PMID:15666807

  12. UniProt Knowledgebase: a hub of integrated protein data

    PubMed Central

    Magrane, Michele; Consortium, UniProt

    2011-01-01

    The UniProt Knowledgebase (UniProtKB) acts as a central hub of protein knowledge by providing a unified view of protein sequence and functional information. Manual and automatic annotation procedures are used to add data directly to the database while extensive cross-referencing to more than 120 external databases provides access to additional relevant information in more specialized data collections. UniProtKB also integrates a range of data from other resources. All information is attributed to its original source, allowing users to trace the provenance of all data. The UniProt Consortium is committed to using and promoting common data exchange formats and technologies, and UniProtKB data is made freely available in a range of formats to facilitate integration with other databases. Database URL: http://www.uniprot.org/ PMID:21447597

  13. UniProt Knowledgebase: a hub of integrated protein data.

    PubMed

    Magrane, Michele

    2011-01-01

    The UniProt Knowledgebase (UniProtKB) acts as a central hub of protein knowledge by providing a unified view of protein sequence and functional information. Manual and automatic annotation procedures are used to add data directly to the database while extensive cross-referencing to more than 120 external databases provides access to additional relevant information in more specialized data collections. UniProtKB also integrates a range of data from other resources. All information is attributed to its original source, allowing users to trace the provenance of all data. The UniProt Consortium is committed to using and promoting common data exchange formats and technologies, and UniProtKB data is made freely available in a range of formats to facilitate integration with other databases. Database URL: http://www.uniprot.org/ PMID:21447597

  14. The Protein Information Resource: an integrated public resource of functional annotation of proteins

    PubMed Central

    Wu, Cathy H.; Huang, Hongzhan; Arminski, Leslie; Castro-Alvear, Jorge; Chen, Yongxing; Hu, Zhang-Zhi; Ledley, Robert S.; Lewis, Kali C.; Mewes, Hans-Werner; Orcutt, Bruce C.; Suzek, Baris E.; Tsugita, Akira; Vinayaka, C. R.; Yeh, Lai-Su L.; Zhang, Jian; Barker, Winona C.

    2002-01-01

    The Protein Information Resource (PIR) serves as an integrated public resource of functional annotation of protein data to support genomic/proteomic research and scientific discovery. The PIR, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the PIR-International Protein Sequence Database (PSD), the major annotated protein sequence database in the public domain, containing about 250 000 proteins. To improve protein annotation and the coverage of experimentally validated data, a bibliography submission system is developed for scientists to submit, categorize and retrieve literature information. Comprehensive protein information is available from iProClass, which includes family classification at the superfamily, domain and motif levels, structural and functional features of proteins, as well as cross-references to over 40 biological databases. To provide timely and comprehensive protein data with source attribution, we have introduced a non-redundant reference protein database, PIR-NREF. The database consists of about 800 000 proteins collected from PIR-PSD, SWISS-PROT, TrEMBL, GenPept, RefSeq and PDB, with composite protein names and literature data. To promote database interoperability, we provide XML data distribution and open database schema, and adopt common ontologies. The PIR web site (http://pir.georgetown.edu/) features data mining and sequence analysis tools for information retrieval and functional identification of proteins based on both sequence and annotation information. The PIR databases and other files are also available by FTP (ftp://nbrfa.georgetown.edu/pir_databases). PMID:11752247

  15. Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery

    NASA Astrophysics Data System (ADS)

    Matsubayashi, Hideaki; Kuruma, Yutetsu; Ueda, Takuya

    2014-12-01

    The cell membrane has many indispensable functions for sustaining cell alive besides a role as merely outer envelope. The most of such functions are implemented by membrane embedded proteins that are emerged through the membrane integration machinery, SecYEG translocon. Here, we synthesized SecYEG by expressing the corresponding gene in vitro to study the process of functionalization of the cell membrane.

  16. Research Resource: Monitoring Endoplasmic Reticulum Membrane Integrity in β-Cells at the Single-Cell Level

    PubMed Central

    Kanekura, Kohsuke; Ou, Jianhong; Hara, Takashi; Zhu, Lihua J.

    2015-01-01

    Endoplasmic reticulum (ER) membrane integrity is an emerging target for human chronic diseases associated with ER stress. Despite the underlying importance of compromised ER membrane integrity in disease states, the entire process leading to ER membrane permeabilization and cell death is still not clear due to technical limitations. Here we describe a novel method for monitoring ER membrane integrity at the single-cell level in real time. Using a β-cell line expressing ER-targeted redox sensitive green fluorescent protein, we could identify a β-cell population undergoing ER membrane permeabilization induced by palmitate and could monitor cell fate and ER stress of these cells at the single-cell level. Our method could be used to develop a novel therapeutic modality targeting the ER membrane for ER-associated disorders, including β-cell death in diabetes, neurodegeneration, and Wolfram syndrome. PMID:25584413

  17. InterPro: the integrative protein signature database

    PubMed Central

    Hunter, Sarah; Apweiler, Rolf; Attwood, Teresa K.; Bairoch, Amos; Bateman, Alex; Binns, David; Bork, Peer; Das, Ujjwal; Daugherty, Louise; Duquenne, Lauranne; Finn, Robert D.; Gough, Julian; Haft, Daniel; Hulo, Nicolas; Kahn, Daniel; Kelly, Elizabeth; Laugraud, Aurélie; Letunic, Ivica; Lonsdale, David; Lopez, Rodrigo; Madera, Martin; Maslen, John; McAnulla, Craig; McDowall, Jennifer; Mistry, Jaina; Mitchell, Alex; Mulder, Nicola; Natale, Darren; Orengo, Christine; Quinn, Antony F.; Selengut, Jeremy D.; Sigrist, Christian J. A.; Thimma, Manjula; Thomas, Paul D.; Valentin, Franck; Wilson, Derek; Wu, Cathy H.; Yeats, Corin

    2009-01-01

    The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or ‘signatures’ representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total ∼58 000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein–protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/). PMID:18940856

  18. PathPPI: an integrated dataset of human pathways and protein-protein interactions.

    PubMed

    Tang, HaiLin; Zhong, Fan; Liu, Wei; He, FuChu; Xie, HongWei

    2015-06-01

    Integration of pathway and protein-protein interaction (PPI) data can provide more information that could lead to new biological insights. PPIs are usually represented by a simple binary model, whereas pathways are represented by more complicated models. We developed a series of rules for transforming protein interactions from pathway to binary model, and the protein interactions from seven pathway databases, including PID, BioCarta, Reactome, NetPath, INOH, SPIKE and KEGG, were transformed based on these rules. These pathway-derived binary protein interactions were integrated with PPIs from other five PPI databases including HPRD, IntAct, BioGRID, MINT and DIP, to develop integrated dataset (named PathPPI). More detailed interaction type and modification information on protein interactions can be preserved in PathPPI than other existing datasets. Comparison analysis results indicate that most of the interaction overlaps values (O AB) among these pathway databases were less than 5%, and these databases must be used conjunctively. The PathPPI data was provided at http://proteomeview.hupo.org.cn/PathPPI/PathPPI.html. PMID:25591449

  19. Thermodynamics of protein destabilization in live cells

    PubMed Central

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

    2015-01-01

    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. PMID:26392565

  20. Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering.

    PubMed

    Boari de Lima, Elisa; Meira, Wagner; Melo-Minardi, Raquel Cardoso de

    2016-06-01

    As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem's complexity. Hence, this work's purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting

  1. Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering

    PubMed Central

    Boari de Lima, Elisa; Meira, Wagner; de Melo-Minardi, Raquel Cardoso

    2016-01-01

    As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem’s complexity. Hence, this work’s purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting

  2. A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

    PubMed Central

    Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

    2014-01-01

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. PMID:25486605

  3. Transparent antennas for solar cell integration

    NASA Astrophysics Data System (ADS)

    Yasin, Tursunjan

    Transparent patch antennas are microstrip patch antennas that have a certain level of optical transparency. Highly transparent patch antennas are potentially suitable for integration with solar panels of small satellites, which are becoming increasingly important in space exploration. Traditional patch antennas employed on small satellites compete with solar cells for surface area. However, a transparent patch antenna can be placed directly on top of solar cells and resolve the issue of competing for limited surface real estate. For such an integration, a high optical transparency of the patch antenna is required from the solar cells' point of view. On the other hand, the antenna should possess at least acceptable radiation properties at the same time. This dissertation focuses on some of the most important concerns from the perspective of small satellite applications. For example, an optimization method to simultaneously improve both optical transparency and radiation efficiency of the antenna is studied. Active integrated antenna design method is extended to meshed patch applications in an attempt to improve the overall power efficiency of the front end communication subsystem. As is well known, circular polarization is immune from Faraday rotation effect in the ionosphere and thus can avoid a 3-dB loss in geo-satellite communication. Therefore, this research also aims to present design methods for circularly polarized meshed patch antennas. Moreover, a meshed patch antenna capable of supporting a high communication data rate is investigated. Lastly, other types of transparent patch antennas are also analyzed and compared to meshed patches. In summary, many properties of transparent patch antennas are examined in order to meet different design requirements.

  4. Rationally designed logic integration of regulatory signals in mammalian cells

    NASA Astrophysics Data System (ADS)

    Leisner, Madeleine; Bleris, Leonidas; Lohmueller, Jason; Xie, Zhen; Benenson, Yaakov

    2010-09-01

    Molecular-level information processing is essential for `smart' in vivo nanosystems. Natural molecular computing, such as the regulation of messenger RNA (mRNA) synthesis by special proteins called transcription factors, has inspired engineered systems that can control the levels of mRNA with certain combinations of transcription factors. Here, we show an alternative approach to achieving general-purpose control of mRNA and protein levels by logic integration of transcription factor input signals in mammalian cells. The transcription factors regulate synthetic genes coding for small regulatory RNAs (called microRNAs), which, in turn, control the mRNA of interest (the output) via an RNA interference pathway. The simplicity of these modular interactions makes it possible, in theory, to implement any arbitrary logic relation between the transcription factors and the output. We construct, test and optimize increasingly complex circuits with up to three transcription factor inputs, establishing a platform for in vivo molecular computing.

  5. Value-added food: single cell protein.

    PubMed

    Anupama; Ravindra, P

    2000-10-01

    The alarming rate of population growth has increased the demand for food production in third-world countries leading to a yawning gap in demand and supply. This has led to an increase in the number of hungry and chronically malnourished people. This situation has created a demand for the formulation of innovative and alternative proteinaceous food sources. Single cell protein (SCP) production is a major step in this direction. SCP is the protein extracted from cultivated microbial biomass. It can be used for protein supplementation of a staple diet by replacing costly conventional sources like soymeal and fishmeal to alleviate the problem of protein scarcity. Moreover, bioconversion of agricultural and industrial wastes to protein-rich food and fodder stocks has an additional benefit of making the final product cheaper. This would also offset the negative cost value of wastes used as substrate to yield SCP. Further, it would make food production less dependent upon land and relieve the pressure on agriculture. This article reviews diversified aspects of SCP as an alternative protein-supplementing source. Various potential strains and substrates that could be utilized for SCP production are described. Nutritive value and removal of nucleic acids and toxins from SCP as a protein-supplementing source are discussed. New processes need to be exploited to improve yield. In that direction the solid state fermentation (SSF) method and its advantages for SCP production are highlighted. PMID:14538097

  6. Mechanisms of integral membrane protein insertion and folding

    PubMed Central

    2014-01-01

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. PMID:25277655

  7. Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)*

    PubMed Central

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

    2015-01-01

    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. PMID:25666614

  8. Sertoli cells secrete both testis-specific and serum proteins.

    PubMed Central

    Wright, W W; Musto, N A; Mather, J P; Bardin, C W

    1981-01-01

    The secretions of the Sertoli cell were examined with two polyvalent antisera--one prepared against proteins in rat serum and the other against testis-specific proteins in rete testis fluid. These antisera detected 12 serum and 9 testis-specific proteins in rete testis fluid. To determine the origin of these proteins, primary cultures enriched in Sertoli cells were incubated with [35S]methionine, and the radiolabeled proteins in the medium were immunoprecipitated. Gel electrophoresis of the two immunoprecipitates resolved eight serum and nine testis-specific proteins. These two sets of proteins were specifically bound to their respective antiserum and were immunologically distinct. Medium from Sertoli cell cultures contained 10 times more of the testis-specific proteins than did cultures enriched for testicular myoid or interstitial cells. The concentration of the serum proteins in Sertoli cell medium was 5 and 10 times greater, respectively, than in myoid or interstitial cell preparations. The proteins from Sertoli cells were next characterized on two-dimensional gels. Seven of the proteins recognized by antiserum against serum proteins had identical molecular weights and isoelectric points as serum proteins. Three of these proteins were ceruloplasmin, transferrin, and glycoprotein 2. In addition to the proteins immunoprecipitated by the two antisera, more than 60 other proteins were detected on two-dimensional gels of the total secretory proteins. We conclude that the Sertoli cell secretes many proteins, some of which are specific to the testis and others of which are similar to serum proteins. Images PMID:6950398

  9. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    NASA Astrophysics Data System (ADS)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  10. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier.

    PubMed

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  11. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    PubMed Central

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  12. Highly efficient single cell arraying by integrating acoustophoretic cell pre-concentration and dielectrophoretic cell trapping.

    PubMed

    Kim, Soo Hyeon; Antfolk, Maria; Kobayashi, Marina; Kaneda, Shohei; Laurell, Thomas; Fujii, Teruo

    2015-11-21

    To array rare cells at the single-cell level, the volumetric throughput may become a bottleneck in the cell trapping and the subsequent single-cell analysis, since the target cells per definition commonly exist in a large sample volume after purification from the original sample. Here, we present a novel approach for high throughput single cell arraying by integrating two original microfluidic devices: an acoustofluidic chip and an electroactive microwell array. The velocity of the cells is geared down in the acoustofluidic chip while maintaining a high volume flow rate at the inlet of the microsystem, and the cells are subsequently trapped one by one into the microwell array using dielectrophoresis. The integrated system exhibited a 10 times improved sample throughput compared to trapping with the electroactive microwell array chip alone, while maintaining a highly efficient cell recovery above 90%. The results indicate that the serial integration of the acoustophoretic pre-concentration with the dielectrophoretic cell trapping drastically improves the performance of the electroactive microwell array for highly efficient single cell analysis. This simple and effective system for high throughput single cell arraying with further possible integration of additional functions, including cell sorting and downstream analysis after cell trapping, has potential for development to a highly integrated and automated platform for single-cell analysis of rare cells. PMID:26439940

  13. Gene stability in mammalian cells and protein consistency.

    PubMed

    Berthold, W

    1994-01-01

    The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well. PMID:7883100

  14. Lists2Networks: Integrated analysis of gene/protein lists

    PubMed Central

    2010-01-01

    Background Systems biologists are faced with the difficultly of analyzing results from large-scale studies that profile the activity of many genes, RNAs and proteins, applied in different experiments, under different conditions, and reported in different publications. To address this challenge it is desirable to compare the results from different related studies such as mRNA expression microarrays, genome-wide ChIP-X, RNAi screens, proteomics and phosphoproteomics experiments in a coherent global framework. In addition, linking high-content multilayered experimental results with prior biological knowledge can be useful for identifying functional themes and form novel hypotheses. Results We present Lists2Networks, a web-based system that allows users to upload lists of mammalian genes/proteins onto a server-based program for integrated analysis. The system includes web-based tools to manipulate lists with different set operations, to expand lists using existing mammalian networks of protein-protein interactions, co-expression correlation, or background knowledge co-annotation correlation, as well as to apply gene-list enrichment analyses against many gene-list libraries of prior biological knowledge such as pathways, gene ontology terms, kinase-substrate, microRNA-mRAN, and protein-protein interactions, metabolites, and protein domains. Such analyses can be applied to several lists at once against many prior knowledge libraries of gene-lists associated with specific annotations. The system also contains features that allow users to export networks and share lists with other users of the system. Conclusions Lists2Networks is a user friendly web-based software system expected to significantly ease the computational analysis process for experimental systems biologists employing high-throughput experiments at multiple layers of regulation. The system is freely available at http://www.lists2networks.org. PMID:20152038

  15. Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors.

    PubMed

    Flower, Darren R; Attwood, Teresa K

    2004-12-01

    G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein. The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics. In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods. BLAST and FastA offer broad brush strokes. Motif-based search methods add the fine detail. Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding. Together, these different views provide a more informative and a more detailed picture of GPCR structure and function. Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow. PMID:15561589

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

    PubMed Central

    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

  17. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  18. Cell surface receptors for CCN proteins.

    PubMed

    Lau, Lester F

    2016-06-01

    The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities. PMID:27098435

  19. High-throughput protein analysis integrating bioinformatics and experimental assays.

    PubMed

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

  20. TeloPIN: a database of telomeric proteins interaction network in mammalian cells.

    PubMed

    Luo, Zhenhua; Dai, Zhiming; Xie, Xiaowei; Feng, Xuyang; Liu, Dan; Songyang, Zhou; Xiong, Yuanyan

    2015-01-01

    Interaction network surrounding telomeres has been intensively studied during the past two decades. However, no specific resource by integrating telomere interaction information data is currently available. To facilitate the understanding of the molecular interaction network by which telomeres are associated with biological process and diseases, we have developed TeloPIN (Telomeric Proteins Interaction Network) database (http://songyanglab.sysu.edu.cn/telopin/), a novel database that points to provide comprehensive information on protein-protein, protein-DNA and protein-RNA interaction of telomeres. TeloPIN database contains four types of interaction data, including (i) protein--protein interaction (PPI) data, (ii) telomeric proteins ChIP-seq data, (iii) telomere-associated proteins data and (iv) telomeric repeat-containing RNAs (TERRA)-interacting proteins data. By analyzing these four types of interaction data, we found that 358 and 199 proteins have more than one type of interaction information in human and mouse cells, respectively. We also developed table browser and TeloChIP genome browser to help researchers with better integrated visualization of interaction data from different studies. The current release of TeloPIN database includes 1111 PPI, eight telomeric protein ChIP-seq data sets, 1391 telomere-associated proteins and 183 TERRA-interacting proteins from 92 independent studies in mammalian cells. The interaction information provided by TeloPIN database will greatly expand our knowledge of telomeric proteins interaction network. PMID:25792605

  1. The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

    PubMed

    Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R; Dutch, Rebecca E; Wei, Yinan

    2016-04-26

    ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro. PMID:27078234

  2. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-01-01

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines. PMID:27573113

  3. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

    PubMed

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R; Fox, Paul L; Gerson, Stanton L; Hoppel, Charles L; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. PMID:26595448

  4. Generation of Mouse Induced Pluripotent Stem Cells by Protein Transduction

    PubMed Central

    Nemes, Csilla; Varga, Eszter; Polgar, Zsuzsanna; Klincumhom, Nuttha; Pirity, Melinda K.

    2014-01-01

    Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription–polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with

  5. Enhanced protein fold recognition through a novel data integration approach

    PubMed Central

    2009-01-01

    Background Protein fold recognition is a key step in protein three-dimensional (3D) structure discovery. There are multiple fold discriminatory data sources which use physicochemical and structural properties as well as further data sources derived from local sequence alignments. This raises the issue of finding the most efficient method for combining these different informative data sources and exploring their relative significance for protein fold classification. Kernel methods have been extensively used for biological data analysis. They can incorporate separate fold discriminatory features into kernel matrices which encode the similarity between samples in their respective data sources. Results In this paper we consider the problem of integrating multiple data sources using a kernel-based approach. We propose a novel information-theoretic approach based on a Kullback-Leibler (KL) divergence between the output kernel matrix and the input kernel matrix so as to integrate heterogeneous data sources. One of the most appealing properties of this approach is that it can easily cope with multi-class classification and multi-task learning by an appropriate choice of the output kernel matrix. Based on the position of the output and input kernel matrices in the KL-divergence objective, there are two formulations which we respectively refer to as MKLdiv-dc and MKLdiv-conv. We propose to efficiently solve MKLdiv-dc by a difference of convex (DC) programming method and MKLdiv-conv by a projected gradient descent algorithm. The effectiveness of the proposed approaches is evaluated on a benchmark dataset for protein fold recognition and a yeast protein function prediction problem. Conclusion Our proposed methods MKLdiv-dc and MKLdiv-conv are able to achieve state-of-the-art performance on the SCOP PDB-40D benchmark dataset for protein fold prediction and provide useful insights into the relative significance of informative data sources. In particular, MKLdiv-dc further

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

    SciTech Connect

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

    1987-02-10

    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 ..cap alpha.. 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 ..beta.. subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the ..cap alpha.. 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 s..cap alpha../ relative to G/sub ichemically bond/ and G/sub ochemically bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with (/sup 125/I)protein. Immunohistochemical studies using an antiserum against the ..beta.. 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 ..cap alpha.. 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.

  7. The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

    PubMed

    Baturin, Simon; Galka, Jamie J; Piyadasa, Hadeesha; Gajjeraman, S; O'Neil, Joe D

    2014-12-01

    Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed. PMID:25387032

  8. Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools.

    PubMed

    Balasubramanian, Sowmya; Matasci, Mattia; Kadlecova, Zuzana; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2015-04-20

    Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3-4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins. PMID:25758242

  9. Reverse phase protein arrays in signaling pathways: a data integration perspective

    PubMed Central

    Creighton, Chad J; Huang, Shixia

    2015-01-01

    The reverse phase protein array (RPPA) data platform provides expression data for a prespecified set of proteins, across a set of tissue or cell line samples. Being able to measure either total proteins or posttranslationally modified proteins, even ones present at lower abundances, RPPA represents an excellent way to capture the state of key signaling transduction pathways in normal or diseased cells. RPPA data can be combined with those of other molecular profiling platforms, in order to obtain a more complete molecular picture of the cell. This review offers perspective on the use of RPPA as a component of integrative molecular analysis, using recent case examples from The Cancer Genome Altas consortium, showing how RPPA may provide additional insight into cancer besides what other data platforms may provide. There also exists a clear need for effective visualization approaches to RPPA-based proteomic results; this was highlighted by the recent challenge, put forth by the HPN-DREAM consortium, to develop visualization methods for a highly complex RPPA dataset involving many cancer cell lines, stimuli, and inhibitors applied over time course. In this review, we put forth a number of general guidelines for effective visualization of complex molecular datasets, namely, showing the data, ordering data elements deliberately, enabling generalization, focusing on relevant specifics, and putting things into context. We give examples of how these principles can be utilized in visualizing the intrinsic subtypes of breast cancer and in meaningfully displaying the entire HPN-DREAM RPPA dataset within a single page. PMID:26185419

  10. TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport

    PubMed Central

    Ong, Yan Shan; Tran, Ton Hoai Thi; Gounko, Natalia V.; Hong, Wanjin

    2014-01-01

    ABSTRACT Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206–229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex. PMID:24806965

  11. Regulation of Sp1 by cell cycle related proteins

    PubMed Central

    Tapias, Alicia; Ciudad, Carlos J.; Roninson, Igor B.; Noé, Véronique

    2009-01-01

    Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NFκB inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NFκB had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NFκB, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression. PMID:18769160

  12. STRING v10: protein-protein interaction networks, integrated over the tree of life.

    PubMed

    Szklarczyk, Damian; Franceschini, Andrea; Wyder, Stefan; Forslund, Kristoffer; Heller, Davide; Huerta-Cepas, Jaime; Simonovic, Milan; Roth, Alexander; Santos, Alberto; Tsafou, Kalliopi P; Kuhn, Michael; Bork, Peer; Jensen, Lars J; von Mering, Christian

    2015-01-01

    The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks. PMID:25352553

  13. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

  14. DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

    PubMed

    Kahn, Jason S; Ruiz, Roanna C H; Sureka, Swati; Peng, Songming; Derrien, Thomas L; An, Duo; Luo, Dan

    2016-06-13

    Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 μm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations. PMID:27112709

  15. Synthetic protein interactions reveal a functional map of the cell

    PubMed Central

    Berry, Lisa K; Ólafsson, Guðjón; Ledesma-Fernández, Elena; Thorpe, Peter H

    2016-01-01

    To understand the function of eukaryotic cells, it is critical to understand the role of protein-protein interactions and protein localization. Currently, we do not know the importance of global protein localization nor do we understand to what extent the cell is permissive for new protein associations – a key requirement for the evolution of new protein functions. To answer this question, we fused every protein in the yeast Saccharomyces cerevisiae with a partner from each of the major cellular compartments and quantitatively assessed the effects upon growth. This analysis reveals that cells have a remarkable and unanticipated tolerance for forced protein associations, even if these associations lead to a proportion of the protein moving compartments within the cell. Furthermore, the interactions that do perturb growth provide a functional map of spatial protein regulation, identifying key regulatory complexes for the normal homeostasis of eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.13053.001 PMID:27098839

  16. General Theory for Integrated Analysis of Growth, Gene, and Protein Expression in Biofilms

    PubMed Central

    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. PMID:24376726

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

    PubMed

    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. PMID:26209045

  18. A Measurable Activation of the bZIP Transcription Factor Atf1 in a Fission Yeast Strain Devoid of Stress-activated and Cell Integrity Mitogen-activated Protein Kinase (MAPK) Activities*

    PubMed Central

    Zhou, Xin; Ma, Yan; Kato, Toshiaki; Kuno, Takayoshi

    2012-01-01

    In Schizosaccharomyces pombe, the stress-activated Sty1 MAPK pathway is essential for cell survival under stress conditions. The Sty1 MAPK regulates Atf1 transcription factor to elicit stress responses in extreme conditions of osmolarity and reactive oxygen species-generating agents such as hydrogen peroxide, heat, low glucose, and heavy metal. Herein, using a newly developed Renilla luciferase reporter assay with enhanced detection sensitivity and accuracy, we show that distinct signaling pathways respond to cadmium and other reactive oxygen species-generating agents for the activation of Atf1. Also, surprisingly, a measurable activation of Atf1 transcription factor was still observed devoid of Sty1 MAPK activity. Further genetic and biological analyses revealed that the residual activation is caused by the activation of the cell wall integrity Pmk1 MAPK pathway and a redox-mediated activation of Atf1. PMID:22661707

  19. Systematic identification of signal integration by protein kinase A

    PubMed Central

    Filteau, Marie; Diss, Guillaume; Dubé, Alexandre K.; Schraffl, Andrea; Bachmann, Verena A.; Gagnon-Arsenault, Isabelle; Chrétien, Andrée-Ève; Steunou, Anne-Lise; Dionne, Ugo; Bisson, Nicolas; Stefan, Eduard; Landry, Christian R.

    2015-01-01

    Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein–protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA. PMID:25831502

  20. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:26297486

  2. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  3. An Integrated Metabolic Atlas of Clear Cell Renal Cell Carcinoma.

    PubMed

    Hakimi, A Ari; Reznik, Ed; Lee, Chung-Han; Creighton, Chad J; Brannon, A Rose; Luna, Augustin; Aksoy, B Arman; Liu, Eric Minwei; Shen, Ronglai; Lee, William; Chen, Yang; Stirdivant, Steve M; Russo, Paul; Chen, Ying-Bei; Tickoo, Satish K; Reuter, Victor E; Cheng, Emily H; Sander, Chris; Hsieh, James J

    2016-01-11

    Dysregulated metabolism is a hallmark of cancer, manifested through alterations in metabolites. We performed metabolomic profiling on 138 matched clear cell renal cell carcinoma (ccRCC)/normal tissue pairs and found that ccRCC is characterized by broad shifts in central carbon metabolism, one-carbon metabolism, and antioxidant response. Tumor progression and metastasis were associated with metabolite increases in glutathione and cysteine/methionine metabolism pathways. We develop an analytic pipeline and visualization tool (metabolograms) to bridge the gap between TCGA transcriptomic profiling and our metabolomic data, which enables us to assemble an integrated pathway-level metabolic atlas and to demonstrate discordance between transcriptome and metabolome. Lastly, expression profiling was performed on a high-glutathione cluster, which corresponds to a poor-survival subgroup in the ccRCC TCGA cohort. PMID:26766592

  4. Recombinant protein vaccines produced in insect cells.

    PubMed

    Cox, Manon M J

    2012-02-27

    The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. PMID:22265860

  5. Proteomic Analysis of Membrane Proteins of Vero Cells: Exploration of Potential Proteins Responsible for Virus Entry

    PubMed Central

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells. PMID:24286161

  6. Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins*

    PubMed Central

    O'Connell, David J.; Bauer, Mikael C.; O'Brien, John; Johnson, Winifred M.; Divizio, Catherine A.; O'Kane, Sara L.; Berggård, Tord; Merino, Alejandro; Åkerfeldt, Karin S.; Linse, Sara; Cahill, Dolores J.

    2010-01-01

    Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca2+ ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca2+. This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (KD ≤ 1 μm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff ≤ 10−3 s−1) and high affinity (KD ≤ 1 μm), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474–493), calmodulin kinase-like vesicle-associated protein (residues 302–316), EF-hand domain family member A2 (residues 202–216), and phosphatidylinositol-4-phosphate 5-kinase, type I, γ (residues 400–415). PMID:20068228

  7. Single-cell protein from waste cellulose

    NASA Technical Reports Server (NTRS)

    Dunlap, C. E.; Callihan, C. D.

    1973-01-01

    The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

  8. Integrated nanoscale tools for interrogating living cells

    NASA Astrophysics Data System (ADS)

    Jorgolli, Marsela

    The development of next-generation, nanoscale technologies that interface biological systems will pave the way towards new understanding of such complex systems. Nanowires -- one-dimensional nanoscale structures -- have shown unique potential as an ideal physical interface to biological systems. Herein, we focus on the development of nanowire-based devices that can enable a wide variety of biological studies. First, we built upon standard nanofabrication techniques to optimize nanowire devices, resulting in perfectly ordered arrays of both opaque (Silicon) and transparent (Silicon dioxide) nanowires with user defined structural profile, densities, and overall patterns, as well as high sample consistency and large scale production. The high-precision and well-controlled fabrication method in conjunction with additional technologies laid the foundation for the generation of highly specialized platforms for imaging, electrochemical interrogation, and molecular biology. Next, we utilized nanowires as the fundamental structure in the development of integrated nanoelectronic platforms to directly interrogate the electrical activity of biological systems. Initially, we generated a scalable intracellular electrode platform based on vertical nanowires that allows for parallel electrical interfacing to multiple mammalian neurons. Our prototype device consisted of 16 individually addressable stimulation/recording sites, each containing an array of 9 electrically active silicon nanowires. We showed that these vertical nanowire electrode arrays could intracellularly record and stimulate neuronal activity in dissociated cultures of rat cortical neurons similar to patch clamp electrodes. In addition, we used our intracellular electrode platform to measure multiple individual synaptic connections, which enables the reconstruction of the functional connectivity maps of neuronal circuits. In order to expand and improve the capability of this functional prototype device we designed

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

    SciTech Connect

    Mushegian, Arcady R.; Elena, Santiago F.

    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.

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

    PubMed

    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. Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

    NASA Astrophysics Data System (ADS)

    Sherman, Paula A.; Fyfe, James A.

    1990-07-01

    The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

  12. Serum proteins are extracted along with monolayer cells in plasticware and interfere with protein analysis

    PubMed Central

    Hong, Xin; Meng, Yuling; Kalkanis, Steven N.

    2016-01-01

    Washing and lysing monolayer cells directly from cell culture plasticware is a commonly used method for protein extraction. We found that multiple protein bands were enriched in samples with low cell numbers from the 6-well plate cultures. These proteins contributed to the overestimation of cell proteins and led to the uneven protein loading in Western blotting analysis. In Coomassie blue stained SDS-PAGE gels, the main enriched protein band is about 69 kDa and it makes up 13.6% of total protein from 104 U251n cells. Analyzed by mass spectrometry, we identified two of the enriched proteins: bovine serum albumin and bovine serum transferrin. We further observed that serum proteins could be extracted from other cell culture plates, dishes and flasks even after washing the cells 3 times with PBS. A total of 2.3 mg of protein was collected from a single well of the 6-well plate. A trace amount of the protein band was still visible after washing the cells 5 times with PBS. Thus, serum proteins should be considered if extracting proteins from plasticware, especially for samples with low cell numbers.

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

    PubMed Central

    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

    2015-01-01

    Summary 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. PMID:26748716

  14. Laser trapping and patterning of protein microcrystals: Toward highly integrated protein microarrays

    NASA Astrophysics Data System (ADS)

    Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Ikeda, Keiko; Shimo-oka, Ai; Mori, Hajime

    2004-09-01

    Some insect virus infections occlude into a crystalline matrix consisting of a protein named polyhedrin. The shape of the matrix is a cubic polyhedron of the size of a few micrometers. Recently it was shown that these polyhedra could immobilize various functional proteins within them. Therefore, the polyhedron is interesting as an element in a protein chip. In this work, individual polyhedra were arrayed and bonded under a microscope by focused laser beams, with the aim of fabricating a highly integrated protein chip. The polyhedron was trapped and transferred to a suitable position on a polymer substrate by optical trapping with a 1064nmNd3+:YAG (YAG, yttrium aluminum garnet) laser. To bond the polyhedron on the substrate, the polymer surface was mechanically and chemically modified by multiphoton absorption of a 120fs, 800nm femtosecond Ti: sapphire laser, which results in strong adhesion of the polyhedron to the substrate. The arraying and bonding of polyhedra were successful, to a precision of about 1μm, with this procedure. The biological activity of polyhedra after these laser irradiations was confirmed by the fluorescence of green fluorescent protein occluded in the polyhedrin matrix.

  15. Integrated Bioinformatics Approach Reveals Crosstalk Between Tumor Stroma and Peripheral Blood Mononuclear Cells in Breast Cancer.

    PubMed

    He, Lang; Wang, Dan; Wei, Na; Guo, Zheng

    2016-01-01

    Breast cancer is now the leading cause of cancer death in women worldwide. Cancer progression is driven not only by cancer cell intrinsic alterations and interactions with tumor microenvironment, but also by systemic effects. Integration of multiple profiling data may provide insights into the underlying molecular mechanisms of complex systemic processes. We performed a bioinformatic analysis of two public available microarray datasets for breast tumor stroma and peripheral blood mononuclear cells, featuring integrated transcriptomics data, protein-protein interactions (PPIs) and protein subcellular localization, to identify genes and biological pathways that contribute to dialogue between tumor stroma and the peripheral circulation. Genes of the integrin family as well as CXCR4 proved to be hub nodes of the crosstalk network and may play an important role in response to stroma-derived chemoattractants. This study pointed to potential for development of therapeutic strategies that target systemic signals travelling through the circulation and interdict tumor cell recruitment. PMID:27039717

  16. Cilostazol strengthens barrier integrity in brain endothelial cells.

    PubMed

    Horai, Shoji; Nakagawa, Shinsuke; Tanaka, Kunihiko; Morofuji, Yoichi; Couraud, Pierre-Oliver; Deli, Maria A; Ozawa, Masaki; Niwa, Masami

    2013-03-01

    We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered. PMID:23224787

  17. SurfaceomeDB: a cancer-orientated database for genes encoding cell surface proteins.

    PubMed

    de Souza, Jorge Estefano Santana; Galante, Pedro Alexandre Favoretto; de Almeida, Renan Valieris Bueno; da Cunha, Julia Pinheiro Chagas; Ohara, Daniel Takatori; Ohno-Machado, Lucila; Old, Lloyd J; de Souza, Sandro José

    2012-01-01

    Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10-20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI. PMID:23390370

  18. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

    Smythe, J.A.; Coppel, R.L.; Brown, G.V.; Ramasamy, R.; Kemp, D.J.; Anders, R.F. )

    1988-07-01

    The authors describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage {lambda}gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a M{sub r} 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a M{sub r} 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with ({sup 3}H)glucosamine and ({sup 3}H)myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the M{sub r} 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

  19. Topological Predictions for Integral Membrane Channel and Carrier Proteins

    PubMed Central

    Abhinay, Reddy; Jaehoon, Cho; Sam, Ling; Vamsee, Reddy; Maksim, Shlykov; Milton, Saier

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS>MEMSAT>HMMTOP>TOPCONS>PHOBIUS>TMHMM>SVMTOP>DAS>S OSUI. Some families, such as the Sugar Porter family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC# 2.A.1) and the Amino acid/Polyamine/Organocation (APC) Family (TC# 2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC# 2.A.29) and the K+ transporter (Trk) families (TC# 2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted poreforming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms 3 were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. PMID:24992992

  20. Integrative approaches for predicting protein function and prioritizing genes for complex phenotypes using protein interaction networks

    PubMed Central

    Ma, Xiaotu; Chen, Ting

    2014-01-01

    With the rapid development of biotechnologies, many types of biological data including molecular networks are now available. However, to obtain a more complete understanding of a biological system, the integration of molecular networks with other data, such as molecular sequences, protein domains and gene expression profiles, is needed. A key to the use of networks in biological studies is the definition of similarity among proteins over the networks. Here, we review applications of similarity measures over networks with a special focus on the following four problems: (i) predicting protein functions, (ii) prioritizing genes related to a phenotype given a set of seed genes that have been shown to be related to the phenotype, (iii) prioritizing genes related to a phenotype by integrating gene expression profiles and networks and (iv) identification of false positives and false negatives from RNAi experiments. Diffusion kernels are demonstrated to give superior performance in all these tasks, leading to the suggestion that diffusion kernels should be the primary choice for a network similarity metric over other similarity measures such as direct neighbors and shortest path distance. PMID:23788799

  1. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    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. PMID:26091838

  2. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  3. 9. Exterior view, Test Cell 7, Systems Integration Laboratory Building ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Exterior view, Test Cell 7, Systems Integration Laboratory Building (T-28), looking southwest. The enclosure discussed in CO-88-B-8 is at the right. - Air Force Plant PJKS, Systems Integration Laboratory, Systems Integration Laboratory Building, Waterton Canyon Road & Colorado Highway 121, Lakewood, Jefferson County, CO

  4. Integrative analysis of cell cycle control in budding yeast.

    PubMed

    Chen, Katherine C; Calzone, Laurence; Csikasz-Nagy, Attila; Cross, Frederick R; Novak, Bela; Tyson, John J

    2004-08-01

    The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo. PMID:15169868

  5. Methods for production of proteins in host cells

    DOEpatents

    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.

  6. Surviving protein quality control catastrophes--from cells to organisms.

    PubMed

    Schneider, Kim; Bertolotti, Anne

    2015-11-01

    Organisms have evolved mechanisms to cope with and adapt to unexpected challenges and harsh conditions. Unfolded or misfolded proteins represent a threat for cells and organisms, and the deposition of misfolded proteins is a defining feature of many age-related human diseases, including the increasingly prevalent neurodegenerative diseases. These protein misfolding diseases are devastating and currently cannot be cured, but are hopefully not incurable. In fact, the aggregation-prone and potentially harmful proteins at the origins of protein misfolding diseases are expressed throughout life, whereas the diseases are late onset. This reveals that cells and organisms are normally resilient to disease-causing proteins and survive the threat of misfolded proteins up to a point. This Commentary will outline the limits of the cellular resilience to protein misfolding, and discuss the possibility of pushing these limits to help cells and organisms to survive the threat of misfolding proteins and to avoid protein quality control catastrophes. PMID:26483388

  7. Biointerface: protein enhanced stem cells binding to implant surface.

    PubMed

    Chrzanowski, W; Kondyurin, A; Lee, Jae Ho; Lord, Megan S; Bilek, M M M; Kim, Hae-Won

    2012-09-01

    The number of metallic implantable devices placed every year is estimated at 3.7 million. This number has been steadily increasing over last decades at a rate of around 8 %. In spite of the many successes of the devices the implantation of biomaterial into tissues almost universally leads to the development of an avascular sac, which consists of fibrous tissue around the device and walls off the implant from the body. This reaction can be detrimental to the function of implant, reduces its lifetime, and necessitates repeated surgery. Clearly, to reduce the number of revision surgeries and improve long-term implant function it is necessary to enhance device integration by modulating cell adhesion and function. In this paper we have demonstrated that it is possible to enhance stem cell attachment using engineered biointerfaces. To create this functional interface, samples were coated with polymer (as a precursor) and then ion implanted to create a reactive interface that aids the binding of biomolecules--fibronectin. Both AFM and XPS analyses confirmed the presence of protein layers on the samples. The amount of protein was significant greater for the ion implanted surfaces and was not disrupted upon washing with detergent, hence the formation of strong bonds with the interface was confirmed. While, for non ion implanted surfaces, a decrease of protein was observed after washing with detergent. Finally, the number of stem cells attached to the surface was enhanced for ion implanted surfaces. The studies presented confirm that the developed bionterface with immobilised fibronectin is an effective means to modulate stem cell attachment. PMID:22714559

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

    PubMed

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

    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. PMID:23369972

  9. Simultaneous extraction of proteins and metabolites from cells in culture.

    PubMed

    Sapcariu, Sean C; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

    2014-01-01

    Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938

  10. Simultaneous extraction of proteins and metabolites from cells in culture

    PubMed Central

    Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

    2014-01-01

    Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology. PMID:26150938