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Sample records for regulates protein turnover

  1. USP21 regulates Hippo pathway activity by mediating MARK protein turnover

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Loya, Anand Chainsukh

    2017-01-01

    observed in cancer and often correlates with worse survival. The activity and stability of Hippo pathway components, including YAP/TAZ, AMOT and LATS1/2, are regulated by ubiquitin-mediated protein degradation. Aberrant expression of ubiquitin ligase complexes that regulate the turnover of Hippo components...

  2. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  3. New aspects of protein stability and turnover in the regulation of genome integrity

    DEFF Research Database (Denmark)

    Gallina, Irene

    of DNA repair is the control of protein abundance, both at a global cellular level, and locally at the site of damage. This is achieved through transcriptional regulation of protein synthesis and through the control of protein stability and turnover. In this study, we investigate the role of Rad56...... sensitivity when mutant. Prior to the work presented here,all these loci have been mapped to a specific gene except RAD56. We map the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in yeast. Deletion of RAD56 causes sensitivity to X-rays, methyl......-scale studies investigating factors involved in DNA metabolism, but no specific function has been assigned to Cmr1. Taking advantage of a series of high-throughput screens we characterize Cmr1 as a chromatinassociated protein, involved in the regulation of fork progression in the presence of replication stress...

  4. Protein turnover in sheep

    International Nuclear Information System (INIS)

    Buttery, P.J.

    1981-01-01

    Considerable advances have been made in the knowledge of the mechanisms and control of synthesis and degradation of proteins in animal tissues during the last decade. Most of the work on the measurement of synthetic and degradative rates of the mixed protein fraction from tissues has been conducted in the rat. There have, unfortunately, been few publications describing results of protein turnover studies with ruminants. Consideration is given here to the techniques used to measure protein turnover, and some of the results obtained, particularly with sheep, are summarized. No attempt has been made to discuss directly the situation in parasitized animals; rather the aim is to provide background information which complements other work dealing with the effects of parasites on the nitrogen metabolism of ruminants. (author)

  5. Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

    DEFF Research Database (Denmark)

    Hagen, Lars; Kavli, Bodil; Sousa, Mirta M L

    2008-01-01

    -catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA......) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23...

  6. Deubiquitylating enzyme USP9x regulates hippo pathway activity by controlling angiomotin protein turnover

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Andrejeva, Diana; Gupta, Rajat

    2016-01-01

    /TAZ activity. We demonstrate that USPx regulates ubiquitin-mediated turnover of the YAP inhibitor, Angiomotin. USP9x acts to deubiquitylate Angiomotin at lysine 496, resulting in stabilization of Angiomotin and lower YAP/TAZ activity. USP9x mRNA levels were reduced in several cancers. Clinically, USP9x m......RNA levels were reduced in several cancers with low USPx expression correlating with poor prognosis in renal clear cell carcinoma. Our data indicate that USP9x may be a useful biomarker for renal clear cell carcinoma....

  7. The glomuvenous malformation protein Glomulin binds Rbx1 and regulates cullin RING ligase-mediated turnover of Fbw7.

    Science.gov (United States)

    Tron, Adriana E; Arai, Takehiro; Duda, David M; Kuwabara, Hiroshi; Olszewski, Jennifer L; Fujiwara, Yuko; Bahamon, Brittany N; Signoretti, Sabina; Schulman, Brenda A; DeCaprio, James A

    2012-04-13

    Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1.

    Science.gov (United States)

    Kang, Shin-Ae; Na, Hyelin; Kang, Hyun-Jin; Kim, Sung-Hye; Lee, Min-Ho; Lee, Mi-Ock

    2010-09-15

    Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein. Copyright 2010 Elsevier Inc. All rights reserved.

  9. Regulation of myofibrillar accumulation in chick muscle cultures - Evidence for the involvement of calcium and lysosomes in non-uniform turnover of contractile proteins

    Science.gov (United States)

    Silver, Geri; Etlinger, Joseph D.

    1985-01-01

    The effects of calcium on the synthesis and the degradation of individual myofibrillar proteins were investigated using primary chick-leg skeletal muscle cultures labeled with S-35-methionine (for protein accumulation experiments) or Ca(2+)-45 (for calcium efflux experiments). It was found that the turnover of individual contractile proteins is regulated nonuniformly by a calcium-dependent mechanism involving lysosomes. The results also indicate that contractile proteins are released from the myofibril before their breakdown to amino acids.

  10. The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+ mouse.

    Directory of Open Access Journals (Sweden)

    James P White

    Full Text Available Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+ mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+ mouse is not known. Cachexia progression was studied in Apc(Min/+ mice that were either weight stable (WS or had initial (≤5%, intermediate (6-19%, or extreme (≥20% body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172, AMPK activity, and raptor phosphorylation (Ser 792 were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.

  11. The Regulation of Skeletal Muscle Protein Turnover during the Progression of Cancer Cachexia in the ApcMin/+ Mouse

    Science.gov (United States)

    White, James P.; Baynes, John W.; Welle, Stephen L.; Kostek, Matthew C.; Matesic, Lydia E.; Sato, Shuichi; Carson, James A.

    2011-01-01

    Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The ApcMin/+ mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the ApcMin/+ mouse is not known. Cachexia progression was studied in ApcMin/+ mice that were either weight stable (WS) or had initial (≤5%), intermediate (6–19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process. PMID:21949739

  12. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  13. The research methods and model of protein turnover in animal

    International Nuclear Information System (INIS)

    Wu Xilin; Yang Feng

    2002-01-01

    The author discussed the concept and research methods of protein turnover in animal body. The existing problems and the research results of animal protein turnover in recent years were presented. Meanwhile, the measures to improve the models of animal protein turnover were analyzed

  14. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison

    2012-03-01

    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  15. Turnover of regulated nurses in long-term care facilities.

    Science.gov (United States)

    Chu, Charlene H; Wodchis, Walter P; McGilton, Katherine S

    2014-07-01

    To describe the relationship between nursing staff turnover in long-term care (LTC) homes and organisational factors consisting of leadership practices and behaviours, supervisory support, burnout, job satisfaction and work environment satisfaction. The turnover of regulated nursing staff [Registered Nurses (RNs) and Registered Practical Nurses (RPNs)] in LTC facilities is a pervasive problem, but there is a scarcity of research examining this issue in Canada. The study was conceptualized using a Stress Process model. Distinct surveys were distributed to administrators to measure organisational factors and to regulated nurses to measure personal and job-related sources of stress and workplace support. In total, 324 surveys were used in the linear regression analysis to examine factors associated with high turnover rates. Higher leadership practice scores were associated with lower nursing turnover; a one score increase in leadership correlated with a 49% decrease in nursing turnover. A significant inverse relationship between leadership turnover and nurse turnover was found: the higher the administrator turnover the lower the nurse turnover rate. Leadership practices and administrator turnover are significant in influencing regulated nurse turnover in LTC. Long-term care facilities may want to focus on building good leadership and communication as an upstream method to minimize nurse turnover. © 2013 John Wiley & Sons Ltd.

  16. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Hübner, Christian A; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and

  17. Regulation of endoplasmic reticulum turnover by selective autophagy

    NARCIS (Netherlands)

    Khaminets, Aliaksandr; Heinrich, Theresa; Mari, Muriel; Grumati, Paolo; Huebner, Antje K.; Akutsu, Masato; Liebmann, Lutz; Stolz, Alexandra; Nietzsche, Sandor; Koch, Nicole; Mauthe, Mario; Katona, Istvan; Qualmann, Britta; Weis, Joachim; Reggiori, Fulvio; Kurth, Ingo; Huebner, Christian A.; Dikic, Ivan

    2015-01-01

    The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements

  18. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover......, there is now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated...... downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work...

  19. Sucrose-phosphate synthase in tree species: light/dark regulation involves a component of protein turnover in Prosopis juliflora (SW DC).

    Science.gov (United States)

    Sinha, A K; Shirke, P A; Pathre, U; Sane, P V

    1997-10-01

    Light dependent modulation of sucrose-phosphate synthase activity (SPS; EC 2.4.1.14) was studied in a tree species, namely Prosopis juliflora. In this paper we demonstrate that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of P. juliflora through transpiration stream in the dark or in light completely prevents in vivo light activation of Vlim and Vmax activities of SPS. In case of spinach, however, cycloheximide feeding affects only Vlim activity while Vmax activity remained unchanged. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplast has no effect on the light activation of SPS in Prosopis. The treatment with cycloheximide showed slight reduction in the rate of O2 evolution indicating that cycloheximide had very little effect on overall photosynthesis. These results indicate that short term protein turnover of the SPS protein and some other essential component(s) (e.g., a putative protein that modifies SPS activity) is one of the primary steps in a complex and unique regulatory cascade effecting the reversible light activation of SPS.

  20. Measurements of protein turnover in man with nitrogen-15

    International Nuclear Information System (INIS)

    Garlick, P.J.; Waterlow, J.C.

    1977-01-01

    Past and present methods of measuring total body protein turnover with 15 N-labelled compounds are reviewed, and a short assessment made of results achieved. Protein turnover in man was first measured by Sprinson and Rittenberg (1949) using 15 N glycine as tracer. The rate of turnover was calculated from the fraction of the dose excreted in urine in the days following a single oral dose of the isotope. In the following years a number of similar methods were devised but interest waned, perhaps because consistent changes in turnover rate under different conditions were not detected. A revival of interest came with the measurement of changes in protein turnover in malnourished and recovering children by Picou and Taylor-Roberts (1969). Nitrogen-15-glycine was infused and measurements were made of the abundance of 15 N in urinary urea, which reached a plateau in about 24 hours. Turnover rates were calculated from the proportion of the infused dose excreted in urea after the plateau had been reached. This method has also been used successfully on adults when the isotope has been given as three-hourly oral doses. In adults, however, plateau labelling in urea is not usually reached for two days. In our laboratory the use of urinary ammonia, which reaches a plateau more quickly than urea, has been examined. In addition, comparisons have been made between constant infusion and single dose of isotope. Halliday and McKeran (1975) infused 15 N-lysine and measured the 15 N abundance in the free lysine of plasma, which reached a plateau in about 12 hours. Turnover in the whole body and turnover of muscle proteins and of plasma albumin were estimated separately. These methods of measuring protein turnover with 15 N are described and results reviewed of the effects of food intake, nutritional status and age. (author)

  1. Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy

    NARCIS (Netherlands)

    de Theije, Chiel C.; Langen, Ramon C. J.; Lamers, Wouter H.; Schols, Annemie M. W. J.; Köhler, S. Eleonore

    2013-01-01

    The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food

  2. Investigation on the turnover of plant proteins. 2

    International Nuclear Information System (INIS)

    Becker, R.; Winkler, E.; Jung, K.; Huebner, G.

    1981-01-01

    Based on kinetic analyses of amino acid and protein turnover by means of compartment models the synthesis and degradation of the soluble proteins in etiolated epicotyl segments of Pisum sativum L. as well as their dependence on the herbicide 2'-methyl-4'-chlorophenoxyacetic acid (MCPA) were determined quantitatively. The segments were incubated for 10 h in a medium containing 14 C-leucine and subsequently placed in an inactive medium. The radioactivity in the soluble proteins and in the amino acid fraction was followed up for a total of 24 h. A 3-pool model in which the total measurable amino acid pool was divided into a direct precursor pool for protein synthesis and into a degradation pool was best suited to interpret the data. The turnover rate for the soluble proteins of untreated epicotyl segments was determined to be 0.058 h -1 ; at an MCPA concentration of 10 -4 M this value was nearly doubled. An increased proteolytic activity in the epicotyl segments ran parallel to the change of the turnover rate in dependence on MCPA concentration. The heterogeneity of the soluble protein with respect to the turnover rate was investigated by means of 3 H/ 14 C double labelling for individual protein fractions separated by gel electrophoresis. The results obtained in this way were comparable with those of the total pool turnover. (author)

  3. Systematic analysis of protein turnover in primary cells.

    Science.gov (United States)

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

    2018-02-15

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

  4. Changes of Protein Turnover in Aging Caenorhabditis elegans

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    Dhondt, Ineke; Petyuk, Vladislav A.; Bauer, Sophie; Brewer, Heather M.; Smith, Richard D.; Depuydt, Geert; Braeckman, Bart P.

    2017-07-05

    Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15Nlabeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, whereas only few proteins show increased turnover. Decrease in protein turnover was consistent for only a minority of functionally related protein subsets, including tubulins and vitellogenins, whereas randomly diverging turnover patterns with age were the norm. Our data suggests increased heterogeneity of protein turnover of the translation machinery, whereas protein turnover of ubiquitin-proteasome and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.

  5. Control of the Protein Turnover Rates in Lemna minor

    Science.gov (United States)

    Trewavas, A.

    1972-01-01

    The control of protein turnover in Lemna minor has been examined using a method described in the previous paper for determining the rate constants of synthesis and degradation of protein. If Lemna is placed on water, there is a reduction in the rate constants of synthesis of protein and an increase (3- to 6-fold) in the rate constant of degradation. The net effect is a loss of protein from the tissue. Omission of nitrate, phosphate, sulfate, magnesium, or calcium results in increases in the rate constant of degradation of protein. An unusual dual effect of benzyladenine on the turnover constants has been observed. Treatment of Lemna grown on sucrose-mineral salts with benzyladenine results in alterations only in the rate constant of synthesis. Treatment of Lemna grown on water with benzyladenine alters only the rate constant of degradation. Abscisic acid on the other hand alters both rate constants of synthesis and degradation of protein together. Inclusion of growth-inhibiting amino acids in the medium results in a reduction in the rate constants of synthesis and increases in the rate constant of degradation of protein. It is concluded that the rate of turnover of protein in Lemna is very dependent on the composition of the growth medium. Conditions which reduce growth rates also reduce the rates of synthesis of protein and increase those of degradation. PMID:16657895

  6. Effects of ractopamine and gender on protein turnover in skeletal ...

    African Journals Online (AJOL)

    Effects of the β-agonist, ractopamine-HCl (ractopamine), on skeletal muscle protein turnover were evaluated in 16 steers (512 kg) and 16 heifers (473 kg). Treatments were arranged in a 2 × 2 factorial design and included gender (steer vs. heifer) and ractopamine (0 or 200 mg/d). Steers were implanted with 120 mg ...

  7. Evolutionary Turnover of Kinetochore Proteins: A Ship of Theseus?

    Science.gov (United States)

    Drinnenberg, Ines A; Henikoff, Steven; Malik, Harmit S

    2016-07-01

    The kinetochore is a multiprotein complex that mediates the attachment of a eukaryotic chromosome to the mitotic spindle. The protein composition of kinetochores is similar across species as divergent as yeast and human. However, recent findings have revealed an unexpected degree of compositional diversity in kinetochores. For example, kinetochore proteins that are essential in some species have been lost in others, whereas new kinetochore proteins have emerged in other lineages. Even in lineages with similar kinetochore composition, individual kinetochore proteins have functionally diverged to acquire either essential or redundant roles. Thus, despite functional conservation, the repertoire of kinetochore proteins has undergone recurrent evolutionary turnover. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

    Science.gov (United States)

    Ishihara, Hirofumi; Moraes, Thiago Alexandre; Pyl, Eva-Theresa; Schulze, Waltraud X; Obata, Toshihiro; Scheffel, André; Fernie, Alisdair R; Sulpice, Ronan; Stitt, Mark

    2017-08-01

    Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO 2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day -1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  9. A high protein diet upregulated whole-body protein turnover during energy deficit

    Science.gov (United States)

    The effects of higher protein diets and sustained energy deficit (ED) on whole-body protein turnover (WBPTO) are not well described. This study examined whether dietary protein level influences whole-body protein breakdown (Ra), non-oxidative leucine disposal (NOLD), and oxidation (Ox) during ED. ...

  10. Feasibility of protein turnover studies in prototroph Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Martin-Perez, Miguel; Villén, Judit

    2015-04-07

    Quantitative proteomics studies of yeast that use metabolic labeling with amino acids rely on auxotrophic mutations of one or more genes on the amino acid biosynthesis pathways. These mutations affect yeast metabolism and preclude the study of some biological processes. Overcoming this limitation, it has recently been described that proteins in a yeast prototrophic strain can also be metabolically labeled with heavy amino acids. However, the temporal profiles of label incorporation under the different phases of the prototroph's growth have not been examined. Labeling trajectories are important in the study of protein turnover and dynamics, in which label incorporation into proteins is monitored across many time points. Here we monitored protein labeling trajectories for 48 h after a pulse with heavy lysine in a yeast prototrophic strain and compared them with those of a lysine auxotrophic yeast. Labeling was successful in prototroph yeast during exponential growth phase but not in stationary phase. Furthermore, we were able to determine the half-lives of more than 1700 proteins during exponential phase of growth with high accuracy and reproducibility. We found a median half-life of 2 h in both strains, which corresponds with the cellular doubling time. Nucleolar and ribosomal proteins showed short half-lives, whereas mitochondrial proteins and other energy production enzymes presented longer half-lives. Except for some proteins involved in lysine biosynthesis, we observed a high correlation in protein half-lives between prototroph and auxotroph strains. Overall, our results demonstrate the feasibility of using prototrophs for proteomic turnover studies and provide a reliable data set of protein half-lives in exponentially growing yeast.

  11. VALSARTAN REGULATES MYOCARDIAL AUTOPHAGY AND MITOCHONDRIAL TURNOVER IN EXPERIMENTAL HYPERTENSION

    Science.gov (United States)

    Zhang, Xin; Li, Zi-Lun; Crane, John A.; Jordan, Kyra L.; Pawar, Aditya S.; Textor, Stephen C.; Lerman, Amir; Lerman, Lilach O.

    2014-01-01

    Renovascular hypertension alters cardiac structure and function. Autophagy is activated during left ventricular hypertrophy and linked to adverse cardiac function. The Angiotensin II receptor blocker Valsartan lowers blood pressure and is cardioprotective, but whether it modulates autophagy in the myocardium is unclear. We hypothesized that Valsartan would alleviate autophagy and improve left ventricular myocardial mitochondrial turnover in swine renovascular hypertension. Domestic pigs were randomized to control, unilateral renovascular hypertension, and renovascular hypertension treated with Valsartan (320 mg/day) or conventional triple therapy (Reserpine+hydralazine+hydrochlorothiazide) for 4 weeks post 6-weeks of renovascular hypertension (n=7 each group). Left ventricular remodeling, function and myocardial oxygenation and microcirculation were assessed by multi-detector computer tomography, blood-oxygen-level-dependent magnetic resonance imaging and microcomputer tomography. Myocardial autophagy, markers for mitochondrial degradation and biogenesis, and mitochondrial respiratory-chain proteins were examined ex vivo. Renovascular hypertension induced left ventricular hypertrophy and myocardial hypoxia, enhanced cellular autophagy and mitochondrial degradation, and suppressed mitochondrial biogenesis. Valsartan and triple therapy similarly decreased blood pressure, but Valsartan solely alleviated left ventricular hypertrophy, ameliorated myocardial autophagy and mitophagy, and increased mitochondrial biogenesis. In contrast, triple therapy only slightly attenuated autophagy and preserved mitochondrial proteins, but elicited no improvement in mitophagy. These data suggest a novel potential role of Valsartan in modulating myocardial autophagy and mitochondrial turnover in renovascular hypertension-induced hypertensive heart disease, which may possibly bolster cardiac repair via a blood pressure-independent manner. PMID:24752430

  12. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A2

    International Nuclear Information System (INIS)

    Burch, R.M.; Axelrod, J.

    1987-01-01

    In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E 2 (PGE 2 ) synthesis. The EC 50 values for stimulation of PGE 2 synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-[γ-thio]triphosphate stimulated PGE 2 synthesis and InsP formation, and guanosine-5'-[β-thio]diphosphate inhibited both PGE 2 synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE 2 synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE 2 synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE 2 synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE 2 synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with [ 3 H] choline, the phospholipase A 2 products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A 2 and that phospholipase A 2 is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis

  13. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  14. Hepatitis C Virus Core Protein Decreases Lipid Droplet Turnover

    Science.gov (United States)

    Harris, Charles; Herker, Eva; Farese, Robert V.; Ott, Melanie

    2011-01-01

    Steatosis is a frequent complication of hepatitis C virus infection. In mice, this condition is recapitulated by the expression of a single viral protein, the nucleocapsid core. Core localizes to the surface of lipid droplets (LDs) in infected liver cells through a process dependent on host diacylglycerol acyltransferase 1 (DGAT1), an enzyme that synthesizes triglycerides in the endoplasmic reticulum. Whether DGAT1 also plays a role in core-induced steatosis is uncertain. Here, we show that mouse embryonic fibroblasts isolated from DGAT1−/− mice are protected from core-induced steatosis, as are livers of DGAT1−/− mice expressing core, demonstrating that the steatosis is DGAT1-dependent. Surprisingly, core expression did not increase DGAT1 activity or triglyceride synthesis, thus excluding the possibility that core activates DGAT1 to cause steatosis. Instead, we find that DGAT1-dependent localization of core to LDs is a prerequisite for the steatogenic properties of the core. Using biochemical and immunofluorescence microscopy techniques, we show that the turnover of lipids in core-coated droplets is decreased, providing a physiological mechanism for core-induced steatosis. Our results support a bipartite model in which core first requires DGAT1 to gain access to LDs, and then LD-localized core interferes with triglyceride turnover, thus stabilizing lipid droplets and leading to steatosis. PMID:21984835

  15. Protein turnover in acid maltase deficiency before and after treatment with a high protein diet.

    OpenAIRE

    Umpleby, A M; Wiles, C M; Trend, P S; Scobie, I N; Macleod, A F; Spencer, G T; Sonksen, P H

    1987-01-01

    A patient with acid maltase deficiency was treated with a high protein diet for 7 months. Protein turnover expressed in terms of lean body mass was shown to be increased in this patient before the diet but was markedly reduced following the diet. The patient improved clinically whilst on the diet both subjectively and in terms of mobility, breathing and reduced peripheral cyanosis at rest.

  16. Turnover of whole body proteins and myofibrillar proteins in middle-aged active men

    International Nuclear Information System (INIS)

    Zackin, M.; Meredith, C.; Frontera, W.; Evans, W.

    1986-01-01

    Endurance-trained older men have a higher proportion of lean tissue and greater muscle cell oxidative capacity, reversing age-related trends and suggesting major changes in protein metabolism. In this study, protein turnover was determined in 6 middle-aged (52+/-1 yr) men who were well trained (VO 2 max 55.2+/-5.0 ml O 2 /kg.min) and lean (body fat 18.9+/-2.8%, muscle mass 36.6+/-0.6%). The maintained habitual exercise while consuming 0.6, 0.9 or 1.2 g protein/kg.day for 10-day periods. N flux was measured from 15 N in urea after oral 15 N-glycine administration. Myofibrillar protein breakdown was estimated from urinary 3-methyl-histidine. Dietary protein had no effect on turnover rates, even when N balance was negative. Whole body protein synthesis was 3.60+/-0.12 g/kg.day and breakdown was 3.40+/-0.14 g/kg.day for all N intakes. Whole body protein flux, synthesis and breakdown were similar to values reported for sedentary young (SY) or sedentary old (SO) men on comparable diets. 3-me-his (3.67+/-0.14 μmol/kg.day) was similar to values reported for SY but higher (p<0.01) than for SO. Myofibrillar protein breakdown per unit muscle mass (185+/-7 μmol 3-me-his/g creatinine) was higher (p<0.01) than for SY or SO. In active middle-aged men, myofibrillar proteins may account for a greater proportion of whole body protein turnover, despite an age-related reduction in muscle mass

  17. Protein turnover and 3-methylhistidine excretion in non-pregnant, pregnant and gestational diabetic women

    International Nuclear Information System (INIS)

    Fitch, W.L.; King, J.C.

    1986-01-01

    Protein turnover was studied in nine non-pregnant (NP) women, eight pregnant (P) and two gestational diabetic (GDM) women. Whole body protein turnover, synthesis and catabolism rates were measured using a single oral dose of 15 N-glycine followed by measurement of enrichment of urinary ammonia. Urinary 3-methylhistidine (3MH) excretion was measured for three consecutive days, including the day of the protein turnover study. Whole body protein turnover and synthesis rates did not differ between the P and NP women, although the synthesis rates tended to be higher in the P group. Gestational diabetic women appeared to have considerably higher rates of both turnover and synthesis. Pregnant women excreted significantly more urinary 3MH than did non-pregnant women. GDM women appeared to have lower 3MH excretion than the P women. Correlation between 3MH excretion and protein turnover rates was nearly significant (p = .06) in the NP women, but was poorly correlated (p = .43) in the P women, suggesting that muscles may be a less important site of whole body protein turnover in pregnancy than in the non-pregnant state

  18. Effect of thyroid hormone on the protein turnover rate of mouse pancreas

    International Nuclear Information System (INIS)

    He Xinjun; Zhou Hui; Wang Shizhen; Zhou Zhonming; Li Liangxue; Wei Huaiwei; Sun Xiaomiao; Wang Yanli

    1986-01-01

    The effects of thyroid hormone on the protein turnover of pancreas in mice were studied using labelled amino acid incorporation, double isotopic and labelled protein decay methods. After injection of L-thyroxine (100 μ g/mouse) for 5 days, the amino acid incorporation into pancreatic proteins of mice was profoundly decreased, the ratio of 3 H/ 14 C in labelled proteins and the fractional turnover rate of pancreatic proteins were also decreased, the protein half-lives being consequently prolonged. These findings suggest that large doses of thyroid hormone may reduce the trunover rate of pancreatic proteins, by inhibiting not only the synthesis but also the degradation

  19. Effect of compounds astragalus extract and cysteamine on finishing pigs growth and protein turnover

    International Nuclear Information System (INIS)

    Wang Cheng; Wang Zhisheng; Zhou Anguo; Liu Guilian; Xue Bai

    2008-01-01

    32 PIC pigs, weighted 59.82±1.32 kg on average, were subjected randomly to 4 treatments for determining the effects of extrogenous metabolism-regulating agents on pig performance, protein turn-over and nitrogen retention by using isotope ( 15 N-Gly) technique and nitrogen balance trial. The 4 treatments were: (1) basal diet; (2) basal diet plus cysteamine (70mg/kg, CS group); (3) basal diet plus non-stable astragalus extracts 250mg/kg (NCAE group); (4) basal diet plus stably astragalus extracts 250mg/kg (CAE group). Astragalus extracts and cysteamine, especially NCAE, redounded to increase pig performance, especially increase average day gain (P<0.05). Both astragalus extracts and cysteamine accelerated protein aggradation rate and amino acid utilization rate, as well as biological value. Astragalus extracts helped to accelerate the synthesis rate of amino acid into body protein and reduce the excretion rate of endogenous urine nitrogen. The mechanism of increasing protein deposition of cysteamine is reducing body protein degradation rate (26.71% reduction, compared to control group), while that of astragalus extracts is bilateral, reducing protein degradation rate (24.84% and 3.66% reduction for NCAE and CAE) and accelerating protein synthesis rate (22.86% and 19.18% increase for NCAE and CAE). Both astragalus extracts and cysteamine contributed the increasing nitrogen retention, net nitrogen utilization and biological value. (authors)

  20. Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

    Science.gov (United States)

    Friel, Claire T; Howard, Jonathon

    2012-12-01

    The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

  1. Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

    International Nuclear Information System (INIS)

    Thoelke, M.S.; Casper, J.M.; Ordal, G.W.

    1990-01-01

    The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it

  2. Regulation of intestinal protein metabolism by amino acids.

    Science.gov (United States)

    Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

    2013-09-01

    Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

  3. Protein Turnover Measurements in Human Serum by Serial Immunoaffinity LC-MS/MS.

    Science.gov (United States)

    Farrokhi, Vahid; Chen, Xiaoying; Neubert, Hendrik

    2018-02-01

    The half-life of target proteins is frequently an important parameter in mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Clinical studies for accurate measurement of physiologically relevant protein turnover can reduce the uncertainty in PK/PD model-based predictions, for example, of the therapeutic dose and dosing regimen in first-in-human clinical trials. We used a targeted mass spectrometry work flow based on serial immunoaffinity enrichment ofmultiple human serum proteins from a [5,5,5- 2 H 3 ]-L-leucine tracer pulse-chase study in healthy volunteers. To confirm the reproducibility of turnover measurements from serial immunoaffinity enrichment, multiple aliquots from the same sample set were subjected to protein turnover analysis in varying order. Tracer incorporation was measured by multiple-reaction-monitoring mass spectrometry and target turnover was calculated using a four-compartment pharmacokinetic model. Five proteins of clinical or therapeutic relevance including soluble tumor necrosis factor receptor superfamily member 12A, tissue factor pathway inhibitor, soluble interleukin 1 receptor like 1, soluble mucosal addressin cell adhesion molecule 1, and muscle-specific creatine kinase were sequentially subjected to turnover analysis from the same human serum sample. Calculated half-lives ranged from 5-15 h; however, no tracer incorporation was observed for mucosal addressin cell adhesion molecule 1. The utility of clinical pulse-chase studies to investigate protein turnover can be extended by serial immunoaffinity enrichment of target proteins. Turnover analysis from serum and subsequently from remaining supernatants provided analytical sensitivity and reproducibility for multiple human target proteins in the same sample set, irrespective of the order of analysis. © 2017 American Association for Clinical Chemistry.

  4. Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

    Science.gov (United States)

    Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

    2013-01-01

    Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load

  5. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  6. Amino acid metabolism and whole-body protein turnover in lambs ...

    African Journals Online (AJOL)

    The effect of protein supplementation of a wheat straw diet on the metabolism of lysine, leucine, methionine and urea, and on whole-body protein turnover rate was investigated in lambs. The metabolism of lysine and leucine is reported elsewhere (Cronje et aI., 1992); in this paper methionine metabolism is discussed, and ...

  7. Determination of protein turnover parameters in athletes using 15N tracers

    International Nuclear Information System (INIS)

    Krumbiegel, P.; Kuehne, K.; Faust, H.; Bornhak, H.; Junghans, P.; Zerbes, H.

    1985-01-01

    In 2 adolescent female athletes engaged in technical-acrobatic sports the influence of a high protein diet on the protein turnover rate under training conditions was investigated by means of 15 N-glycine. Protein synthesis was significantly increased, whereas the utilization of nutritive nitrogen was decreased as expected. The 15 N tracer technique is well suited to determine the protein requirements under special training conditions

  8. Impact of transamination reactions and protein turnover on labeling dynamics in C-13-labeling experiments

    DEFF Research Database (Denmark)

    Grotkjær, Thomas; Åkesson, M.; Christensen, Bjarke

    2004-01-01

    A dynamic model describing carbon atom transitions in the central metabolism of Saccharomyces cerevisiae is used to investigate the influence of transamination reactions and protein turnover on the transient behavior of C-13-labeling chemostat experiments. The simulations performed suggest...... that carbon exchange due to transamination and protein turnover can significantly increase the required time needed for metabolites in the TCA cycle to reach isotopic steady state, which is in agreement with published experimental observations. On the other hand, transamination and protein turnover will speed...... behavior until after three residence times. These observations suggest that greater caution should be used while also pointing to new opportunities in the design and interpretation of C-13-labeling experiments....

  9. Stress and Protein Turnover in Lemna minor1

    Science.gov (United States)

    Cooke, Robert J.; Oliver, Jane; Davies, David D.

    1979-01-01

    Transfer of fronds of Lemna minor L. to adverse growth conditions or stress situations causes a lowering of the growth rate and a loss of soluble protein per frond, the extent of the loss being dependent on the nature of the stress. The loss or protein is due to two factors: (a) a decrease in the rate constant of protein synthesis (ks); (b) an increase in the rate constant of protein degradation (kd). In plants adapted to the stresses, protein synthesis increases and the initially rapid rate of proteolysis is reduced. Addition of abscisic acid both lowers ks and increases kd, whereas benzyladenine seems to alleviate the effects of stress on protein content by decreasing kd rather than by altering ks. Based on the measurement of enzyme activities, stress-induced protein degradation appears to be a general phenomenon, affecting many soluble proteins. The adaptive significance of stress-induced proteolysis is discussed. PMID:16661102

  10. Whole-body protein turnover of a carnivore, Felis silvestris catus.

    Science.gov (United States)

    Russell, K; Lobley, G E; Millward, D J

    2003-01-01

    The cat (Felis silvestris catus) has a higher dietary protein requirement than omnivores and herbivores, thought to be due to metabolic inflexibility. An aspect of metabolic flexibility was examined with studies of whole-body protein turnover at two levels of dietary protein energy, moderate protein (MP; 20 %) and high protein (HP; 70 %), in five adult cats in a crossover design. Following a 14 d pre-feed period, a single intravenous dose of [15N]glycine was administered and cumulative excretion of the isotope in urine and faeces determined over 48 h. N flux increased (Pwork is required to determine exactly why cats have such a high protein requirement.

  11. Influence of Sex and Estrogen on Musculotendinous Protein Turnover at Rest and After Exercise

    DEFF Research Database (Denmark)

    Hansen, Mette; Kjær, Michael

    2014-01-01

    Women differ from men with regard to muscle and tendon, most likely because of sex differences in estrogen. The present experimental findings suggest the hypothesis that estrogen has an anabolic effect on muscle primarily by lowering the protein turnover and enhancing sensitivity to resistance...... training. Furthermore, estrogen may reduce the stiffness of tendons, an effect that may be modified by physical training....

  12. Effects of ractopamine and gender on protein turnover in skeletal ...

    African Journals Online (AJOL)

    p2492989

    Ractopamine significantly decreased rates of in vitro protein degradation ... with 140 mg trenbolone acetate and 14 mg estradiol (Component TE-H, Vet Life). ... samples were not used for statistical analysis, and samples collected after 15 and 29 ..... and its interaction with gender for RNA, DNA, and protein concentrations as.

  13. Development of a method for the accurate measurement of protein turnover in neoplastic cells grown in culture

    International Nuclear Information System (INIS)

    Silverman, J.A.

    1984-01-01

    In this study, it was shown that standard techniques for cell recovery and sample preparation for liquid scintillation counting led to underestimation of the radioactivity present in cell proteins by 20-40%. These techniques involved labeling with 3 He leucine or 14 C leucine, scraping the cells from the dish in a buffer, TCA precipitation of the cell proteins, solubilization in NaOH and counting in a liquid scintillation counter. Hydrolysis of the proteins with HCl or Pronase significantly increased the recovery of the labeled proteins. Also, solubilization in situ with NaOH or hydrolysis in situ with Pronase recovered 5-10% additional labeled proteins. The techniques developed here allow the accurate measurement of radioactivity in cell proteins. In addition, these techniques were used to study protein turnover in rat hepatoma cells grown in culture. These cells regulated their growth rate through changes in the protein synthesis rate as opposed to changes in the protein degradation rate. These data support the hypothesis that neoplastic cells, unlike normal cells, do not regulate proteolysis in growth control; normal cells under similar conditions have been shown to activate lysosomal proteolysis as they reach confluence. The physiologic implications of this observation are discussed

  14. [Legislative regulation of production and turnover of products for people with different diseases].

    Science.gov (United States)

    Pritul'skaia, N V; Motuzka, Iu N; Antiushko, D L

    2013-01-01

    This article presents results of analysis of existing regulatory documents and approaches to the legislative regulation of production and turnover of special dietary products for people with specific diseases in EU, Ukraine and Russian Federation. According to the EU legislation, production and turnover of food products for nutritional support of people during specific diseases and the rehabilitation period are regulated by the Commission Directive 1999/21/EC, 2009/39/ES, by Regulation Commission (EU) No 953/2009 and documents of Codex Committee. Special food products for people with specific diseases in Ukrainian legislation are classified as nutrition products for special dietary use and are regulated by the following Laws of Ukraine "On the safety and quality of food", "On ensuring of sanitary and epidemiological welfare of the population", "On Consumer Rights Protection", "On advertising" and by other non-legislative acts. According to the current legislation of the Russian Federation, the products for people with specific diseases are classified as healthy dietary food products. The basis of the legal framework are federal laws "On the quality and safety of food", "On the sanitary-epidemiological welfare of the populations", "On technical regulations and technical regulations of the Customs Union "On Food Safety" and "On the safety of certain types of specialized food products, including healthy dietary food products and therapeutic dietary food products". There is no common approach to the legal regulation of production and turnover of products for people with specific diseases in the world. The proposals for further harmonization of regulatory control in this area have been developed.

  15. Turnover-dependent inactivation of the nitrogenase MoFe-protein at high pH.

    Science.gov (United States)

    Yang, Kun-Yun; Haynes, Chad A; Spatzal, Thomas; Rees, Douglas C; Howard, James B

    2014-01-21

    Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725-13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis.

  16. Effect of alcohol consumption on whole-body protein turnover in healthy adults.

    Science.gov (United States)

    Wutzke, Klaus D; Krentz, Helga; Bruns, Gerrit

    2011-03-01

    The aim of the study was to investigate the whole-body protein turnover, either before or after continuous, moderate ethanol-induced oxidative stress by red wine consumption over a relatively short period in healthy volunteers. Ten healthy adults received an individual regular diet over 20 days. After 10 days, the subjects consumed 0.4 ml ethanol kg(-1) day(-1) as red wine together with dinner over a 10-day period. After 8 and 18 days, respectively, a (15)N-labelled yeast protein was administered in a dosage of 4.2 mg kg(-1) body weight. Urine and faeces were collected over 48 h, respectively. The (15)N-enrichment was measured by isotope ratio mass spectrometry, whereas the protein flux rates were calculated by a three-compartment model. The whole-body protein turnover without/with red wine consumption amounted to 3.73±0.6 and 3.49±0.6 g kg(-1) day(-1) (not significant), respectively. Moderate alcohol consumption does not induce significant short-term changes in the whole-body protein turnover of healthy adults.

  17. Dystropathology Increases Energy Expenditure and Protein Turnover in the Mdx Mouse Model of Duchenne Muscular Dystrophy

    Science.gov (United States)

    Radley-Crabb, Hannah G.; Marini, Juan C.; Sosa, Horacio A.; Castillo, Liliana I.; Grounds, Miranda D.; Fiorotto, Marta L.

    2014-01-01

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles. PMID:24586653

  18. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  19. In vivo measurement of myocardial protein turnover using an indicator dilution technique

    International Nuclear Information System (INIS)

    Revkin, J.H.; Young, L.H.; Stirewalt, W.S.; Dahl, D.M.; Gelfand, R.A.; Zaret, B.L.; Barrett, E.J.

    1990-01-01

    We applied a nondestructive tracer technique, previously developed for measuring skeletal muscle protein turnover, to the measurement of myocardial protein turnover in vivo. During a continuous infusion of L-[ring-2,6-3H]phenylalanine to anesthetized, overnight-fasted dogs, we measured the uptake of radiolabeled phenylalanine from plasma and the release of unlabeled phenylalanine from myocardial proteolysis using arterial and coronary sinus catheterization and analytic methods previously applied to skeletal muscle. Using these measurements, together with a model of myocardial protein synthesis that assumes rapid equilibration of tracer specific activity between myocardial phenylalanyl-tRNA and circulating phenylalanine, we estimated the rates of heart protein synthesis and degradation. The rate of heart protein synthesis was also estimated directly from the incorporation of labeled phenylalanine into tissue protein. The use of [3H]phenylalanine was compared with L-[1-14C]leucine in the measurement of heart protein turnover in dogs given simultaneous infusion of both tracers. Leucine uptake and release by the myocardium exceeded that of phenylalanine by 3.1 +/- 0.4- and 1.7 +/- 0.3-fold, respectively, consistent with leucine's 2.4-fold greater abundance in heart protein and its metabolism via other pathways. Phenylalanine is the preferred tracer for use with this method because of its limited metabolic fate in muscle. One theoretical limitation to the method, slow equilibration of circulating labeled phenylalanine with myocardial phenylalanyl-tRNA, was resolved by comparison of these specific activities after a 30-minute infusion of labeled phenylalanine in the rat. A second, empirical limitation involves precision in the measurement of the small decrements in phenylalanine specific activity that occur with each pass of blood through the coronary circulation

  20. Casein and soya-bean protein have different effects on whole body protein turnover at the same nitrogen balance

    DEFF Research Database (Denmark)

    Nielsen, K; Kondrup, J; Elsner, Petteri

    1994-01-01

    was recovered from urinary ammonia and urea during isotope steady state for measurement of protein synthesis and protein degradation. Compared with starvation the protein-free diet decreased N excretion by 75%, probably by increasing the rate of reutilization of amino acids from endogenous proteins for protein......The present study examined whether different proteins have different effects on whole-body protein turnover in adult rats. The rats were either starved, given a protein-free but energy-sufficient diet (1 MJ/kg body weight (BW) per d) or a diet containing intact casein, hydrolysed casein......, or hydrolysed soya-bean protein at a level of 9.1 g/kg BW per d. The diets, which were isoenergetic with the same carbohydrate: fat ratio, were given as a continuous intragastric infusion for at least 4 d. During the last 19 h 15N-glycine (a primed continuous infusion) was given intragastrically and 15N...

  1. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    International Nuclear Information System (INIS)

    Poortmans, J.R.; Carpentier, A.; Pereira-Lancha, L.O.; Lancha, A. Jr.

    2012-01-01

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ( 13 C-lysine, 15 N-glycine, 2 H 5 -phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg −1 ·day −1 compared to 0.8 g·kg −1 ·day −1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h

  2. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Energy Technology Data Exchange (ETDEWEB)

    Poortmans, J.R.; Carpentier, A. [Laboratory for Biometry and Sport Nutrition, Faculty of Motor Sciences, Free University of Brussels, Brussels (Belgium); Pereira-Lancha, L.O. [Departamento de Nutrição, Instituto Vita, São Paulo, SP (Brazil); Lancha, A. Jr. [Laboratório de Nutrição Aplicada à Atividade Motora, Escola de Educação Física e Esporte, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-08

    Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers ({sup 13}C-lysine, {sup 15}N-glycine, {sup 2}H{sub 5}-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg{sup −1}·day{sup −1} compared to 0.8 g·kg{sup −1}·day{sup −1} in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  3. Protein turnover, amino acid requirements and recommendations for athletes and active populations

    Directory of Open Access Journals (Sweden)

    J.R. Poortmans

    2012-10-01

    Full Text Available Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes compared to resistance exercise, which induces fiber hypertrophy (myofibrils. Nitrogen balance (difference between protein intake and protein degradation for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins and carbohydrate (± 30 g maltodextrine drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

  4. Effects of dietary protein and glycaemic index on biomarkers of bone turnover in children

    DEFF Research Database (Denmark)

    Dalskov, Stine-Mathilde; Müller, Martha; Ritz, Christian

    2014-01-01

    For decades, it has been debated whether high protein intake compromises bone mineralisation, but no long-term randomised trial has investigated this in children. In the family-based, randomised controlled trial DiOGenes (Diet, Obesity and Genes), we examined the effects of dietary protein...... and glycaemic index (GI) on biomarkers of bone turnover and height in children aged 5-18 years. In two study centres, families with overweight parents were randomly assigned to one of five ad libitum-energy, low-fat (25-30 % energy (E%)) diets for 6 months: low protein/low GI; low protein/high GI; high protein....../low GI; high protein/high GI; control. They received dietary instructions and were provided all foods for free. Children, who were eligible and willing to participate, were included in the study. In the present analyses, we included children with data on plasma osteocalcin or urinary N...

  5. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    interactions with other proteins, or binding of small molecules. Covalent .... vealed through structural elucidation of the protein in free and oxygen-bound forms .... stance, molecular dynamic simulation of glutamine binding pro- tein shows that ...

  6. Interleukin-6 markedly decreases skeletal muscle protein turnover and increases nonmuscle amino acid utilization in healthy individuals

    DEFF Research Database (Denmark)

    van Hall, Gerrit; Steensberg, Adam; Fischer, Christian

    2008-01-01

    CONTEXT: IL-6 is a key modulator of immune function and suggested to be involved in skeletal muscle wasting as seen in sepsis. OBJECTIVE: Our objective was to determine the role of IL-6 in human in vivo systemic and skeletal muscle amino acid metabolism and protein turnover. SUBJECTS AND METHODS...... synthesis was more suppressed than breakdown, causing a small increase in net muscle protein breakdown. Furthermore, rhIL-6 decreased arterial amino acid concentration with 20-40%, despite the increase net release from muscle. CONCLUSIONS: We demonstrated that IL-6 profoundly alters amino acid turnover....... A substantial decrease in plasma amino acids was observed with a concomitant 50% decrease in muscle protein turnover, however, modest increase in net muscle degradation. We hypothesize that the profound reduction in muscle protein turnover and modest increase in net degradation are primarily caused...

  7. Studies on protein turnover and energy expenditure in chronically undernourished adults during stress of infection

    International Nuclear Information System (INIS)

    Kurpad, A.V.; Shetty, P.S.; Reeds, P.J.

    1994-01-01

    Chronic undernutrition in man leads to adaptive responses which could reduce the requirements for dietary energy and protein. It is also possible that these adaptive responses, which are economical in nature, could lead to a decreased capacity for combating stress. Undernourished people are more susceptible to infections, and during these stresses, show different patterns of protein and energy metabolism from well-nourished subjects. Animal models have clearly shown a diminished response to tissue injury, in terms of the anabolic acute phase response. It is proposed to study the effect of prior nutritional status on the degree to which an infective stress stimulates the acute phase protein synthesis by the liver. In addition, the supply of amino acids to the liver in conditions of stress could come from the breakdown of body tissue proteins, particularly muscle. It is intended to study muscle protein turnover by the use of 13 C-leucine in undernourished subjects under conditions of stress. Since whole body protein turnover can be measured by two methods, using 15 N-glycine and 13 C-leucine, a comparison of these two methods will initially be made in chronically undernourished subjects. It is also intended to study daily energy expenditure in the subject by an isotopic method, i.e. the appearance of 13 CO 2 in the breath after the administration of 13 C-bicarbonate. (author). 8 refs

  8. Positive muscle protein net balance and differential regulation of atrogene expression after resistance exercise and milk protein supplementation

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2014-01-01

    Purpose Resistance exercise and amino acid availability are positive regulators of muscle protein net balance (NB). However, anabolic responses to resistance exercise and protein supplementation deserve further elucidation. The purpose was to compare intakes of whey, caseinate (both: 0.30 g/kg lean...... body mass), or a non-caloric control after heavy resistance exercise on protein turnover and mRNA expressions of forkhead homeobox type O (FOXO) isoforms, muscle RING finger 1 (MuRF1), and Atrogin1 in young healthy males. Methods Protein turnover was determined by stable isotope-labeled leucine...

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  10. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    Energy Technology Data Exchange (ETDEWEB)

    Dhondt, Ineke; Petyuk, Vladislav A.; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D.; Depuydt, Geert; Braeckman, Bart P.

    2016-09-01

    Cellular protein quality can be maintained by proteolytic elimination of damaged proteins and replacing them with newly synthesized copies, a process called protein turnover (Ward, 2000). Protein turnover rates have been estimated using SILAC (stable isotope labeling by amino acids in cell culture) in prokaryotes and eukaryotes. The last decade has witnessed a growing interest in the analysis of whole-organism proteome dynamics in metazoans using the same approach (Claydon and Beynon, 2012). In recent work, SILAC was applied to monitor protein synthesis throughout life in adult Caenorhabditis elegans (Vukoti et al., 2015) and to investigate food intake (Gomez-Amaro et al., 2015

  11. Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Tony Ly

    2018-05-01

    Full Text Available Background: Viral oncogenes and mutated proto-oncogenes are potent drivers of cancer malignancy. Downstream of the oncogenic trigger are alterations in protein properties that give rise to cellular transformation and the acquisition of malignant cellular phenotypes. Developments in mass spectrometry enable large-scale, multidimensional characterisation of proteomes. Such techniques could provide an unprecedented, unbiased view of how oncogene activation remodels a human cell proteome. Methods: Using quantitative MS-based proteomics and cellular assays, we analysed how transformation induced by activating v-Src kinase remodels the proteome and cellular phenotypes of breast epithelial (MCF10A cells. SILAC MS was used to comprehensively characterise the MCF10A proteome and to measure v-Src-induced changes in protein abundance across seven time-points (1-72 hrs. We used pulse-SILAC MS (Boisvert et al., 2012, to compare protein synthesis and turnover in control and transformed cells. Follow-on experiments employed a combination of cellular and functional assays to characterise the roles of selected Src-responsive proteins. Results: Src-induced transformation changed the expression and/or turnover levels of ~3% of proteins, affecting ~1.5% of the total protein molecules in the cell. Transformation increased the average rate of proteome turnover and disrupted protein homeostasis. We identify distinct classes of protein kinetics in response to Src activation. We demonstrate that members of the polycomb repressive complex 1 (PRC1 are important regulators of invasion and migration in MCF10A cells. Many Src-regulated proteins are present in low abundance and some are regulated post-transcriptionally. The signature of Src-responsive proteins is highly predictive of poor patient survival across multiple cancer types. Open access to search and interactively explore all these proteomic data is provided via the EPD database (www.peptracker.com/epd. Conclusions

  12. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Cohen, Stephen Michael

    2017-01-01

    /TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family...... deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor...

  13. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans.

    Science.gov (United States)

    Dhondt, Ineke; Petyuk, Vladislav A; Cai, Huaihan; Vandemeulebroucke, Lieselot; Vierstraete, Andy; Smith, Richard D; Depuydt, Geert; Braeckman, Bart P

    2016-09-13

    Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

    Directory of Open Access Journals (Sweden)

    Ineke Dhondt

    2016-09-01

    Full Text Available Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditis elegans and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

  15. The excretion of isotope in urea and ammonia for estimating protein turnover in man with [15N]glycine

    International Nuclear Information System (INIS)

    Fern, E.B.; Garlick, P.J.; McNurlan, M.A.; Waterlow, J.C.

    1981-01-01

    Four normal adults were given [ 15 N]-glycine in a single dose either orally or intravenously. Rates of whole-body protein turnover were estimated from the excretion of 15 N in ammonia and in urea during the following 9 h. The rate derived from urea took account of the [ 15 N]urea retained in body water. In postabsorptive subjects the rates of protein synthesis given by ammonia were equal to those from urea, when the isotope was given orally, but lower when an intravenous dose was given. In subjects receiving equal portions of food every 2 h rates of synthesis calculated from ammonia were much lower than those from urea whether an oral or intravenous isotope was given. Comparison of rates obtained during the postabsorptive and absorptive periods indicated regulation by food intake primarily of synthesis when measurements were made on urea, but regulation primarily of breakdown when measurements were made on ammonia. These inconsistencies suggest that changes in protein metabolism might be assessed better by correlating results given by different end-products, and it is suggested that the mean value given by urea and ammonia will be useful for this purpose. (author)

  16. Excretion of isotope in urea and ammonia for estimating protein turnover in man with (/sup 15/N)glycine

    Energy Technology Data Exchange (ETDEWEB)

    Fern, E B; Garlick, P J; McNurlan, M A; Waterlow, J C [London School of Hygiene and Tropical Medicine (UK)

    1981-01-01

    Four normal adults were given (/sup 15/N)-glycine in a single dose either orally or intravenously. Rates of whole-body protein turnover were estimated from the excretion of /sup 15/N in ammonia and in urea during the following 9 h. The rate derived from urea took account of the (/sup 15/N)urea retained in body water. In postabsorptive subjects the rates of protein synthesis given by ammonia were equal to those from urea, when the isotope was given orally, but lower when an intravenous dose was given. In subjects receiving equal portions of food every 2 h rates of synthesis calculated from ammonia were much lower than those from urea whether an oral or intravenous isotope was given. Comparison of rates obtained during the postabsorptive and absorptive periods indicated regulation by food intake primarily of synthesis when measurements were made on urea, but regulation primarily of breakdown when measurements were made on ammonia. These inconsistencies suggest that changes in protein metabolism might be assessed better by correlating results given by different end-products, and it is suggested that the mean value given by urea and ammonia will be useful for this purpose.

  17. Resistance training reduces whole-body protein turnover and improves net protein retention in untrained young males.

    Science.gov (United States)

    Hartman, Joseph W; Moore, Daniel R; Phillips, Stuart M

    2006-10-01

    It is thought that resistance exercise results in an increased need for dietary protein; however, data also exists to support the opposite conclusion. The purpose of this study was to determine the impact of resistance exercise training on protein metabolism in novices with the hypothesis that resistance training would reduce protein turnover and improve whole-body protein retention. Healthy males (n = 8, 22 +/- 1 y, BMI = 25.3 +/- 1.8 kg.m(-2)) participated in a progressive whole-body split routine resistance-training program 5d/week for 12 weeks. Before (PRE) and after (POST) the training, oral [15N]-glycine ingestion was used to assess nitrogen flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NPB = PS-PB). Macronutrient intake was controlled over a 5d period PRE and POST, while estimates of protein turnover and urinary nitrogen balance (N(bal) = N(in) - urine N(out)) were conducted. Bench press and leg press increased 40% and 50%, respectively (p training-induced increases in both NPB (PRE = 0.22 +/- 0.13 g.kg(-1).d(-1); POST = 0.54 +/- 0.08 g.kg(-1).d(-1)) and urinary nitrogen balance (PRE = 2.8 +/- 1.7 g N.d(-1); POST = 6.5 +/- 0.9 g N.d(-1)) were observed. A program of resistance training that induced significant muscle hypertrophy resulted in reductions of both whole-body PS and PB, but an improved NPB, which favoured the accretion of skeletal muscle protein. Urinary nitrogen balance increased after training. The reduction in PS and PB and a higher NPB in combination with an increased nitrogen balance after training suggest that dietary requirements for protein in novice resistance-trained athletes are not higher, but lower, after resistance training.

  18. Olfactory marker protein: turnover and transport in normal and regenerating neurons

    International Nuclear Information System (INIS)

    Kream, R.M.; Margolis, F.L.

    1984-01-01

    A 19,000-dalton acidic protein designated olfactory marker protein (OMP) is a cell-specific marker of mature olfactory chemosensory neurons. Intranasal irrigation of mouse olfactory epithelium with [ 35 S]methionine labeled OMP to high specific activity. Turnover and transport characteristics of 35 S-labeled OMP were compared to those of 35 S-labeled global cytosol protein in groups of young, adult, and Triton-treated adult mice. The latter contained primarily large numbers of regenerating olfactory neurons. In olfactory epithelium of young and Triton-treated mice, the specific activity of OMP was three times that of global cytosol protein, whereas in adults the two measures were equal. In all three groups, however, the rate of degradation of OMP was roughly equal to that of cytosol protein (T1/2 . 5 to 6 days). By contrast, differences in T1/2 for OMP decline in the bulb of adult, young, and Triton-treated adult mice were highly significant (T1/2's of 9.3, 6.1, and 4 to 5 days, respectively; p . 0.001). The specific activity of [35S]methionine incorporated in OMP exceeded that of the free amino acid 5-fold, indicating minimal precursor reutilization during the course of our experiments. Turnover data indicate that increased isotope incorporation into OMP in the epithelium is matched by an accelerated rate of degradation in the bulb. This may be correlated with the physiological state or developmental age of the primary neurons since in young and Triton-treated adult mice, rapidly maturing ''young'' olfactory neurons represent a larger proportion of the total population than in adults. Thus, OMP behaves as a typical, relatively slowly transported soluble protein (v . 2 to 4 mm/day, slow component b)

  19. The ways to improve state regulation of land turnover which are in a government and municipal property in Ukraine

    Directory of Open Access Journals (Sweden)

    S. A. Serdyuk

    2016-09-01

    Full Text Available After comprehensive scientific exploration the ways of improvement of state regulation of land turnover which are in a government and state municipal property in Ukraine have been identified. Also it was proposed and justified the conceptual bases of strategy in development of state regulation. As a result of scientific exploration the directions of improvement of organizational mechanism have been formed also and wich a viev to improve legal mechanism of state regulation of land turnover it was suggested the modern approach of it. The main problem that constrains mechanical development of state regulation is non-compliance with legal principles, corruption and low motivation of officials. Approach to improvement is necessity of strategic development by the way of compliance law principles, realization of identified steps in development, using the practice of international regulation in land turnover. Strategic goals are in improvement of procedure of land turnover in public property by the way of advance mechanisms of state regulation; in a finding of effective approaches to improvement components of the legal mechanism; to produce and to offer propositions about development of components legal mechanism; to create clear and understandable procedure of land turnover for all participants in relationships in accordance with European legal traditions etc.Scenario of development that was elected is strategic and directed to solve the problems, perfection of economic, organizational and legal mechanisms of land turnover and also system development. But there are some risks that can interfere realization of strategic priorities. They are in outside threat, political instability, the economic downturn and corruption. We have chosen the next evaluation criteria: the level of investment attractiveness; the level of perception corruption in the society; the level of rating and satisfying quality of in land turnover process by the market participants with

  20. Turnover of amyloid precursor protein family members determines their nuclear signaling capability.

    Science.gov (United States)

    Gersbacher, Manuel T; Goodger, Zoë V; Trutzel, Annette; Bundschuh, Diana; Nitsch, Roger M; Konietzko, Uwe

    2013-01-01

    The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, β-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65.

  1. Associations of total, dairy, and meat protein with markers for bone turnover in healthy, prepubertal boys

    DEFF Research Database (Denmark)

    Budek, Alicja Zofia; Hoppe, Camilla; Michaelsen, Kim Fleischer

    2007-01-01

    intake was estimated from a 3-d weighed food record. sIGF-I and its binding protein-3 were assessed (immunoassay) in a subgroup of 56 boys. All statistical models included effects of age, BMI, and energy intake. Dairy protein was negatively associated with sOC (P ¼ 0.05) but not significantly associated......We previously reported that high intake of milk, but not meat, equal in protein content, increased serum insulin-like growth factor-I (sIGF-I) in prepubertal boys. sIGF-I plays a key role in bone metabolism. Therefore, the aim of this cross-sectional study was to investigate associations of total.......04) but not significantly associated with sOC and sCTX. Free sIGF-I was positively associated with total (P , 0.01) and dairy (P ¼ 0.06) protein but not with meat protein. Our results indicate that dairy and meat protein may exhibit a distinct regulatory effect on different markers for bone turnover. Future studies should...

  2. Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature.

    Science.gov (United States)

    Kucerová, H; Strnadová, M; Ludvík, J; Chaloupka, J

    1999-01-01

    In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C). Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5. Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the

  3. Whole-body protein turnover response to short-term high-protein diets during weight loss: a randomized controlled trial

    Science.gov (United States)

    Objective: Determine whole-body protein turnover responses to high protein diets during weight loss. Design: Thirty-nine adults (age, 21 ± 1 yr; VO2peak, 48 ± 1 ml'kg-1'min-1; body mass index, 25 ± 1 kg•m2) were randomized to diets providing protein at the recommend dietary allowance (RDA), 2X-RD...

  4. Protein turnover in malnourished patients with cystic fibrosis: Effects of elemental and nonelemental nutritional supplements

    International Nuclear Information System (INIS)

    Pelekanos, J.T.; Holt, T.L.; Ward, L.C.; Cleghorn, G.J.; Shepherd, R.W.

    1990-01-01

    To evaluate the relative efficacy of nonelemental versus semielemental enteral supplements for nutritional rehabilitation of cystic fibrosis (CF) patients, whole-body protein turnover using the [ 15 N]glycine method was studied in nine malnourished CF patients during enteral feedings, in a block design study comparing a semielemental formula (Criticare), a higher protein density but nonelemental formula (Traumacal) (T), and a nonelemental formula that had been modified to become isocaloric and isonitrogenous to the semielemental formula (modified Traumacal, MT). No significant differences in rates of protein synthesis or catabolism were observed comparing the three formulas. However, the higher protein density nonelemental formula resulted in higher net protein deposition compared to the other two formulas (T + 0.42 g kg-1 10 h-1 versus 0.33 g kg-1 10 h-1 for Criticare and -0.59 g kg-1 10 h-1 for MT), although this was significant (p less than 0.05) for the MT versus T comparison only. This study lends support to the use of less expensive nonelemental formulas for the nutritional management of malnourished patients with CF

  5. Active Gating, Molecular Pumping, and Turnover Determination in Biomimetic Lipidic Cubic Mesophases with Reconstituted Membrane Proteins.

    Science.gov (United States)

    Speziale, Chiara; Zabara, Alexandru Florian; Drummond, Calum John; Mezzenga, Raffaele

    2017-11-28

    Understanding the mechanisms controlling molecular transport in bioinspired materials is a central topic in many branches of nanotechnology. In this work, we show that biomolecules of fundamental importance in biological processes, such as glucose, can be transported in an active, controlled, and selective manner across macroscopic lipidic cubic mesophases, by correctly reconstituting within them their corresponding membrane protein transporters, such as Staphylococcus epidermidis (GlcP Se ). Importantly, by duly exploiting the symporter properties of GlcP Se of coupled glucose/H + transport, the diffusion of glucose can further be tuned by independent physiological stimuli, such as parallel or antiparallel pH gradients, offering an important model to study molecular exchange processes in cellular machinery. We finally show that by measuring the transport properties of the lipidic mesophases with and without the GlcP Se membrane protein reconstituted within, it becomes possible to determine its intrinsic conductance. We generalize these findings to other membrane proteins from the antiporters family, such as the bacterial ClC exchanger from Escherichia coli (EcClC), providing a robust method for evaluating the turnover rate of the membrane proteins in general.

  6. Studies on the possible role of thyroid hormone in altered muscle protein turnover during sepsis

    International Nuclear Information System (INIS)

    Hasselgren, P.O.; Chen, I.W.; James, J.H.; Sperling, M.; Warner, B.W.; Fischer, J.E.

    1987-01-01

    Five days after thyroidectomy (Tx) or sham-Tx in young male Sprague-Dawley rats, sepsis was induced by cecal ligation and puncture (CLP). Control animals underwent laparotomy and manipulation of the cecum without ligation or puncture. Sixteen hours after CLP or laparotomy, protein synthesis and degradation were measured in incubated extensor digitorum longus (EDL) and soleus (SOL) muscles by determining rate of 14 C-phenylalanine incorporation into protein and tyrosine release into incubation medium, respectively. Triiodothyronine (T3) was measured in serum and muscle tissue. Protein synthesis was reduced by 39% and 22% in EDL and SOL, respectively, 16 hours after CLP in sham-Tx rats. The response to sepsis of protein synthesis was abolished in Tx rats. Protein breakdown was increased by 113% and 68% in EDL and SOL, respectively, 16 hours after CLP in sham-Tx animals. The increase in muscle proteolysis during sepsis was blunted in hypothyroid animals and was 42% and 49% in EDL and SOL, respectively. T3 in serum was reduced by sepsis, both in Tx and sham-Tx rats. T3 in muscle, however, was maintained or increased during sepsis. Abolished or blunted response of muscle protein turnover after CLP in hypothyroid animals may reflect a role of thyroid hormones in altered muscle protein metabolism during sepsis. Reduced serum levels of T3, but maintained or increased muscle concentrations of the hormone, suggests that increased T3 uptake by muscle may be one mechanism of low T3 syndrome in sepsis, further supporting the concept of a role for thyroid hormone in metabolic alterations in muscle during sepsis

  7. Blood protein turnover in parasitized ruminants. The influence of host nutrition

    International Nuclear Information System (INIS)

    Dargie, J.D.

    1981-01-01

    Ruminants infected with helminth or protozoal parasites generally become anaemic and hypoalbuminaemic, as well as losing their appetite. Since feed intake plays an important part in determining blood protein levels, it is necessary, when attempting to determine the mechanisms by which parasites cause anaemia and hypoalbuminaemia, to differentiate between the effects of feed intake per se and the specific effects of the parasite on blood protein turnover. This can be done by a variety of radioisotope techniques using infected and pair-fed control animals. Additionally, animals on a poor plane of nutrition suffer more from parasitism than those which are well fed. To understand the reason for this, it is necessary to determine whether diet influences susceptibility to parasite establishment or survival, and/or susceptibility to the metabolic consequences of parasitism. Described here is the current state of knowledge on the interaction between host nutrition and susceptibility to parasitic infection and parasitic disease processes, with particular reference to anaemia and hypoalbuminaemia. It is concluded that there is little evidence that nutrition has a significant bearing on resistance or susceptibility to infection, but that it does not have a profound influence on the ability of animals to withstand the pathogenic effects of parasites. The reasons for this are discussed in detail, but the principal benefit of a good plane of nutrition is that it enables the synthetic machinery of the host to keep pace with the concurrent parasite-induced hypercatabolism of blood proteins. (author)

  8. Protein turnover in lactating mink (Mustela vison) is not affected by dietary protein supply

    DEFF Research Database (Denmark)

    Tauson, Anne-Helene; Fink, Rikke; Chwalibog, André

    2006-01-01

    The mink is a strict carnivore and may therefore serve as a model for the cat. Current recommendations for protein supply for lactating mink are based on production experiments with preweaning kit growth as a measure of dietary adequacy (1,2). Recently, nitrogen balance and substrate oxidation have...... in humans (7), growing pigs (8), and growing rats (9). In adult cats, both protein synthesis and breakdown were lower when feeding a low- than when feeding a high-protein diet [20 vs. 70% of metabolizable energy (ME)5 from protein] (10). The objectives of this study were therefore to develop a ¹5N...

  9. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

    Directory of Open Access Journals (Sweden)

    MALAV SUCHIN TRIVEDI

    2012-06-01

    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  10. Effects of resistance training and protein supplementation on bone turnover in young adult women

    Directory of Open Access Journals (Sweden)

    Sinning Wayne E

    2005-08-01

    Full Text Available Abstract Background The strength of aging bone depends on the balance between the resorption and formation phases of the remodeling process. The purpose of this study was to examine the interaction of two factors with the potential to exert opposing influences on bone turnover, resistance exercise training and high dietary protein intake. It was hypothesized that resistance training by young, healthy, untrained women with protein intakes near recommended levels (0.8 g·kg-1·d-1 would promote bone formation and/or inhibit bone resorption, and that subsequent supplementation to provide 2.4 g protein·kg-1·d-1 would reverse these effects. Methods Bone formation was assessed with serum bone-specific alkaline phosphatase (BAP and osteocalcin (OC, and bone resorption with urinary calcium and deoxypyridinoline (DPD. Biochemical, strength, anthropometric, dietary, and physical activity data were obtained from 24 healthy, untrained, eumenorrheic women (18–29y at baseline, after eight weeks of resistance training (3 d·wk-1, ~1 hr·d-1; 3 sets, 6–10 repetitions, 13 exercises, 75–85% maximum voluntary contraction, and after 12 weeks of resistance training and 10 days of protein/placebo supplementation. Subjects were randomized (double-blind to either a high protein (HP or training control (TC group and, during the final 10 days, consumed either enough purified whey protein to bring daily protein intake to 2.4 g·kg-1·d-1, or an equivalent dose of isoenergetic, carbohydrate placebo. Results Strength, lean tissue mass, and DPD increased significantly in both groups over time, while percent body fat and BAP decreased (repeated measures ANOVA, p ≤ 0.05, Bonferroni correction. No significant changes were observed for serum OC or urinary calcium, and no significant group (TC, HP × time (baseline, week 8, week 12 interactions emerged for any of the biochemical measures. Conclusion (1 Twelve weeks of high-intensity resistance training did not appear to

  11. Oncogenic pathways and myrna: effects on messenger's turnover, regulation by synthetic oligo ribonucleotides and therapeutic applications in experimental settings

    International Nuclear Information System (INIS)

    Nicolin, A.

    2009-01-01

    The project has been focusing on molecular mechanisms determining the rate of RNA degradation eventually important to regulate gene expression and phenotype at post transcription level. Exogenous synthetic oligonucleotides targeting the relevant domains of b- RNA degradation could stabilize the transcript, efficiently enhance gene expression and alter the cellular phenotype accordingly. The experimental model was the bcl2 RNA (b-RNA), its molecular mechanisms of degradation and the functional effects of turnover modifications by exogenous means

  12. Radiocarbon Dating of the Human Eye Lens Crystallines Reveal Proteins without Carbon Turnover throughout Life

    Science.gov (United States)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen; Jacobsen, Christina; Heinemeier, Jan

    2008-01-01

    Background Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule) completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its 14C content from the atmospheric carbon dioxide. The 14C content of the lens proteins thus reflects the atmospheric content of 14C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the 14C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric 14C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating. Methodology/Principal Findings Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation. Conclusions/Significance Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the 14C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA). Potential targets may be nervous tissues in terms of senile or pre-senile degradation, as well as other highly

  13. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life.

    Directory of Open Access Journals (Sweden)

    Niels Lynnerup

    Full Text Available BACKGROUND: Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14C content from the atmospheric carbon dioxide. The (14C content of the lens proteins thus reflects the atmospheric content of (14C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the (14C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric (14C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating. METHODOLOGY/PRINCIPAL FINDINGS: Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation. CONCLUSIONS/SIGNIFICANCE: Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the (14C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA. Potential targets may be nervous tissues in terms of senile or pre

  14. Acute moderate elevation of TNF-{alpha} does not affect systemic and skeletal muscle protein turnover in healthy humans

    DEFF Research Database (Denmark)

    Petersen, Anne Marie; Plomgaard, Peter; Fischer, Christian P

    2009-01-01

    -alpha infusion (rhTNF-alpha). We hypothesize that TNF-alpha increases human muscle protein breakdown and/or inhibit synthesis. Subjects and Methods: Using a randomized controlled, crossover design post-absorptive healthy young males (n=8) were studied 2 hours under basal conditions followed by 4 hours infusion...... with the phenylalanine 3-compartment model showed similar muscle synthesis, breakdown and net muscle degradation after 2 hours basal and after 4 hours Control or rhTNF-alpha infusion. Conclusion: This study is the first to show in humans that TNF-alpha does not affect systemic and skeletal muscle protein turnover, when......Context: Skeletal muscle wasting has been associated with elevations in circulating inflammatory cytokines, in particular TNF-alpha. Objective: In this study, we investigated whether TNF-alpha affects human systemic and skeletal muscle protein turnover, via a 4 hours recombinant human TNF...

  15. Effects of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle as measured by [14C]tyrosine infusion.

    OpenAIRE

    Carter, W J; Benjamin, W S; Faas, F H

    1982-01-01

    The effect of T3 (3,3',5-tri-iodothyronine) on protein turnover in skeletal and cardiac muscle was measured in intact rats by means of a 6 h [14C]tyrosine-infusion technique. Treatment with 25-30 micrograms of T3/100 g body wt. daily for 4-7 days increased the fractional rate of protein synthesis in skeletal muscle. Since the fractional growth rate of the muscle was decreased or unchanged, T3 treatment increased the rate of muscle protein breakdown. These findings suggest that increased prote...

  16. Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid.

    Science.gov (United States)

    Tsai, Shang-Yi A; Pokrass, Michael J; Klauer, Neal R; Nohara, Hiroshi; Su, Tsung-Ping

    2015-05-26

    Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

  17. The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Wen

    Full Text Available The HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4 to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2 and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1. DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.We found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4(DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

  18. Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

    Science.gov (United States)

    Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

    2014-01-01

    During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

  19. Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover

    International Nuclear Information System (INIS)

    Ju, H.-J.; Ye, C.-M.; Verchot-Lubicz, Jeanmarie

    2008-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocate to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway

  20. TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly

    Science.gov (United States)

    Uchil, Pradeep D.; Pawliczek, Tobias; Reynolds, Tracy D.; Ding, Siyuan; Hinz, Angelika; Munro, James B.; Huang, Fang; Floyd, Robert W.; Yang, Haitao; Hamilton, William L.; Bewersdorf, Joerg; Xiong, Yong; Calderwood, David A.; Mothes, Walther

    2014-01-01

    ABSTRACT Focal adhesions are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of focal adhesions is crucial for cell migration. Paxillin is a multi-adaptor protein that plays an important role in regulating focal adhesion dynamics. Here, we identify TRIM15, a member of the tripartite motif protein family, as a paxillin-interacting factor and a component of focal adhesions. TRIM15 localizes to focal contacts in a myosin-II-independent manner by an interaction between its coiled-coil domain and the LD2 motif of paxillin. Unlike other focal adhesion proteins, TRIM15 is a stable focal adhesion component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and reduced focal adhesion disassembly rates, in addition to enlarged focal adhesions. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of focal adhesion turnover and cell migration. PMID:25015296

  1. Effects of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle as measured by [14C]tyrosine infusion.

    Science.gov (United States)

    Carter, W J; Benjamin, W S; Faas, F H

    1982-04-15

    The effect of T3 (3,3',5-tri-iodothyronine) on protein turnover in skeletal and cardiac muscle was measured in intact rats by means of a 6 h [14C]tyrosine-infusion technique. Treatment with 25-30 micrograms of T3/100 g body wt. daily for 4-7 days increased the fractional rate of protein synthesis in skeletal muscle. Since the fractional growth rate of the muscle was decreased or unchanged, T3 treatment increased the rate of muscle protein breakdown. These findings suggest that increased protein degradation is an important factor in decreasing skeletal-muscle mass in hyperthyroidism. In contrast with skeletal muscle, T3 treatment for 7 days caused an equivalent increase in the rate of cardiac muscle growth and protein synthesis. This suggests that hyperthyroidism does not increase protein breakdown in heart muscle as it does in skeletal muscle. The failure of T3 to increase proteolysis in heart muscle may be due to a different action on the cardiac myocyte or to systemic effects of T3 which increase cardiac work.

  2. Peptide Level Turnover Measurements Enable the Study of Proteoform Dynamics.

    Science.gov (United States)

    Zecha, Jana; Meng, Chen; Zolg, Daniel Paul; Samaras, Patroklos; Wilhelm, Mathias; Kuster, Bernhard

    2018-05-01

    The coordination of protein synthesis and degradation regulating protein abundance is a fundamental process in cellular homeostasis. Today, mass spectrometry-based technologies allow determination of endogenous protein turnover on a proteome-wide scale. However, standard dynamic SILAC (Stable Isotope Labeling in Cell Culture) approaches can suffer from missing data across pulse time-points limiting the accuracy of such analysis. This issue is of particular relevance when studying protein stability at the level of proteoforms because often only single peptides distinguish between different protein products of the same gene. To address this shortcoming, we evaluated the merits of combining dynamic SILAC and tandem mass tag (TMT)-labeling of ten pulse time-points in a single experiment. Although the comparison to the standard dynamic SILAC method showed a high concordance of protein turnover rates, the pulsed SILAC-TMT approach yielded more comprehensive data (6000 proteins on average) without missing values. Replicate analysis further established that the same reproducibility of turnover rate determination can be obtained for peptides and proteins facilitating proteoform resolved investigation of protein stability. We provide several examples of differentially turned over splice variants and show that post-translational modifications can affect cellular protein half-lives. For example, N-terminally processed peptides exhibited both faster and slower turnover behavior compared with other peptides of the same protein. In addition, the suspected proteolytic processing of the fusion protein FAU was substantiated by measuring vastly different stabilities of the cleavage products. Furthermore, differential peptide turnover suggested a previously unknown mechanism of activity regulation by post-translational destabilization of cathepsin D as well as the DNA helicase BLM. Finally, our comprehensive data set facilitated a detailed evaluation of the impact of protein

  3. RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Andreea Iren Serban

    Full Text Available AGEs accumulation in the skin affects extracellular matrix (ECM turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

  4. BAR domain proteins regulate Rho GTPase signaling.

    Science.gov (United States)

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

  5. Regulation of cardiac C-protein phosphorylation

    International Nuclear Information System (INIS)

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

  6. The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Murcha, Monika W; Jańska, Hanna

    2018-01-29

    Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i -AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i -AAA protease in the regulation of mitochondrial biogenesis in plants. © 2018. Published by The Company of Biologists Ltd.

  7. Evidence for the Deregulation of Protein Turnover Pathways in Atm-Deficient Mouse Cerebellum: An Organotypic Study.

    Science.gov (United States)

    Kim, Catherine D; Reed, Ryan E; Juncker, Meredith A; Fang, Zhide; Desai, Shyamal D

    2017-07-01

    Interferon-stimulated gene 15 (ISG15), an antagonist of the ubiquitin pathway, is elevated in cells and brain tissues obtained from ataxia telangiectasia (A-T) patients. Previous studies reveal that an elevated ISG15 pathway inhibits ubiquitin-dependent protein degradation, leading to activation of basal autophagy as a compensatory mechanism for protein turnover in A-T cells. Also, genotoxic stress (ultraviolet [UV] radiation) deregulates autophagy and induces aberrant degradation of ubiquitylated proteins in A-T cells. In the current study, we show that, as in A-T cells, ISG15 protein expression is elevated in cerebellums and various other tissues obtained from Atm-compromised mice in an Atm-allele-dependent manner (Atm+/+ Atm+/- Atm-/-). Notably, in cerebellums, the brain part primarily affected in A-T, levels of ISG15 were significantly greater (3-fold higher) than cerebrums obtained from the same set of mice. Moreover, as in A-T cell culture, UV induces aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient, but not in Atm-proficient, cerebellar brain slices grown in culture. Thus, the ex vivo organotypic A-T mouse brain culture model mimics that of an A-T human cell culture model and could be useful for studying the role of ISG15-dependent proteinopathy in cerebellar neurodegeneration, a hallmark of A-T in humans. © 2017 American Association of Neuropathologists, Inc. All rights reserved.

  8. Anchoring Proteins as Regulators of Signaling Pathways

    Science.gov (United States)

    Perino, Alessia; Ghigo, Alessandra; Scott, John D.; Hirsch, Emilio

    2012-01-01

    Spatial and temporal organization of signal transduction is coordinated through the segregation of signaling enzymes in selected cellular compartments. This highly evolved regulatory mechanism ensures the activation of selected enzymes only in the vicinity of their target proteins. In this context, cAMP-responsive triggering of protein kinase A is modulated by a family of scaffold proteins referred to as A-kinase anchoring proteins. A-kinase anchoring proteins form the core of multiprotein complexes and enable simultaneous but segregated cAMP signaling events to occur in defined cellular compartments. In this review we will focus on the description of A-kinase anchoring protein function in the regulation of cardiac physiopathology. PMID:22859670

  9. Muscle protein turnover in the elderly and its potential contribution to the development of sarcopenia.

    Science.gov (United States)

    Murton, Andrew J

    2015-11-01

    The underlying aetiology of sarcopenia appears multifaceted and not yet fully defined, but ultimately involves the gradual loss of muscle protein content over time. The present evidence suggests that the loss of lean tissue in the elderly is exacerbated by low dietary protein intake. Moreover, acute stable-isotope-based methodologies have demonstrated that the muscle anabolic response to a given amount of protein may decline with age, a phenomenon that has been termed anabolic resistance. Although the mechanism responsible for the inability of muscle to mount a satisfactory anabolic response to protein provision with increasing age is presently unknown, it does not appear due to impaired digestion or absorption of dietary protein. Rather, the issue could reside with any combination of: a diminished delivery of amino acids to peripheral tissues, impaired uptake of amino acids into muscle cells, or an inability of amino acids to elicit intracellular events pivotal for anabolism to occur. Despite the presence of anabolic resistance to dietary protein, present evidence suggests that protein supplementation may be able to overcome these issues, particularly when combined with resistance exercise programmes. As such, protein supplementation may prove to be an effective approach to delay the loss of muscle mass with age and has led to calls for the recommended daily intake of protein to be increased for the elderly population.

  10. Regulation and Turnover of Nitric Oxide by Phytoglobins in Plant Cell Responses

    DEFF Research Database (Denmark)

    Igamberdiev, Abir U; Hebelstrup, Kim; Stasolla, Claudio

    2016-01-01

    The involvement of phytoglobins in the metabolism of nitric oxide (NO) and reactive nitrogen species (RNS) produced during stress, plant growth, and development is discussed. The action of phytoglobin expression upon NO leads to the maintenance of redox status, minimization of the damage from...... to the mobility of both NO and phytohormones, plants developed strategies to regulate specific cell hormonal actions to permit differentiation during development and to respond to stress. Phytoglobins are the agents responsible for differential cellular responses to hormones that use NO as a signal transduction...... reactive oxygen and nitrogen species in the cytoplasm of the cell, and regulation of hormonal and stress responses. NO scavenging is achieved via phytoglobins, and it can also involve S-nitrosoglutathione reductase and a direct interaction of NO with superoxide anion followed by detoxification of formed...

  11. Muscle protein turnover in cattle of differing genetic backgrounds as measured by urinary N tau-methylhistidine excretion

    International Nuclear Information System (INIS)

    McCarthy, F.D.; Bergen, W.G.; Hawkins, D.R.

    1983-01-01

    N tau-methylhistidine (N tau MH) was used as an index for muscle protein degradation and this index was utilized to evaluate degradation rates in young growing cattle. Initially, two Charolais crossbred heifers, 12 months of age, were used to measure the recovery of radioactivity in the urine for a 120-hour period after intravenous injection of [ 14 C]N tau MH. Of the radioactivity injected into the animals, 89.7% was recovered after 120 hours. With rate and amount of clearance as the criteria, the excretion of N tau MH in urine appears to be a valid index of muscle protein degradation in cattle. Eight steers of two genetic types were used to evaluate the effect of frame size on turnover rates of muscle proteins with N tau MH as an index. Large frame cattle (LG) excreted more N tau MH per day throughout the trial. Total daily creatinine excretion was less for small frame (SM) cattle showing an increase with time in LG and SM cattle. N tau MH-to-creatinine ratios showed a decline with time. Fractional breakdown rates (FBR) and fractional synthesis rates (FSR) appeared to parallel each other with rates tending to decrease with age. No differences were observed between LG and SM cattle for FBR, FSR or fractional growth rate

  12. Protein turnover and metabolism in the elderly intensive care unit patient

    NARCIS (Netherlands)

    Phillips, Stuart M.; Dickerson, Roland N.; Moore, Frederick A.; Paddon-Jones, Douglas; Weijs, Peter J. M.

    2017-01-01

    Many intensive care unit (ICU) patients do not achieve target protein intakes particularly in the early days following admittance. This period of iatrogenic protein undernutrition contributes to a rapid loss of lean, in particular muscle, mass in the ICU. The loss of muscle in older (aged >60 years)

  13. Transcriptional regulation by Polycomb group proteins

    DEFF Research Database (Denmark)

    Di Croce, Luciano; Helin, Kristian

    2013-01-01

    Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins......) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity....

  14. Studies on water turnover and water requirement in buffalo calves fed on different levels of energy and protein in the diet

    International Nuclear Information System (INIS)

    Verma, D.N.; Kumar, N.; Dass, R.S.; Singh, U.B.

    1980-01-01

    Experiments were conducted for measuring water turnover and water requirement in buffalo calves fed on different levels of energy and protein in the diet. There was significant difference (P<0.01) in the biological half life of tritiated water between various groups of animals. Water turnover rates were statistically (P<0.05) more in groups I and II (106.52 and 99.74 ml/kg/24 hr) than groups III and IV (93.03 and 77.09 ml/kg/24 hr). There was no significant difference in water requirement due to various treatments. (author)

  15. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Chris Snowdon

    Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  16. Naturally occurring stable isotopes reflect changes in protein turnover and growth in gilthead sea bream (Sparus aurata) juveniles under different dietary protein levels.

    Science.gov (United States)

    Martin-Perez, Miguel; Fernandez-Borras, Jaume; Ibarz, Antoni; Felip, Olga; Fontanillas, Ramon; Gutierrez, Joaquim; Blasco, Josefina

    2013-09-18

    Ideal nutritional conditions are crucial to sustainable aquaculture due to economic and environmental issues. Here we apply stable isotope analysis as an indicator of fish growth and feeding balance, to define the optimum diet for efficient growing conditions. Juveniles of gilthead sea bream were fed with six isoenergetic diets differing in protein to lipid proportion (from 41/26 to 57/20). As protein intake increased, δ¹⁵N and Δδ¹⁵N of muscle and Δδ¹⁵N and Δδ¹³C of its protein fraction decreased, indicating lower protein turnover and higher protein deposition in muscle. This is reflected in the inverse relationship found between Δδ¹⁵N and growth rate, although no differences were observed in either parameter beyond the protein/lipid proportion 47/23. Principal component analysis (PCA) also signaled 47/23 diet as the pivotal point with the highest growing efficiency, with isotopic parameters having the highest discrimination load. Thus, muscle isotope composition, especially ¹⁵N, can be used to evaluate nutritional status in farmed fish.

  17. Effect of experimental hyperthyroidism on protein turnover in skeletal and cardiac muscle.

    Science.gov (United States)

    Carter, W J; Van Der Weijden Benjamin, W S; Faas, F H

    1980-10-01

    Since experimental hyperthyroidism reduces skeletal muscle mass while simultaneously increasing cardiac muscle mass, the effect of hyperthyroidism on muscle protein degradation was compared in skeletal and cardiac muscle. Pulse-labeling studies using (3H) leucine and (14C) carboxyl labeled aspartate and glutamate were carried out. Hyperthyroidism caused a 25%-29% increase in protein breakdown in both sarcoplasmic and myofibrillar fractions of skeletal muscle. Increased muscle protein degradation may be a major factor in the development of skeletal muscle wasting and weakness in hyperthyroidism. In contrast, protein breakdown appeared to be reduced 22% in the sarcoplasmic fraction of hyperthyroid heart muscle and was unchanged in the myofibrillar fraction. Possible reasons for the contrasting effects of hyperthyroidism on skeletal and cardiac muscle include increased sensitivity of the hyperthyroid heart to catecholamines, increased cardiac work caused by the hemodynamic effects of hyperthyroidism, and a different direct effect of thyroid hormone at the nuclear level in cardiac as opposed to skeletal muscle.

  18. Efficacy of Corrected Rapid Turnover Protein Increment Index (CRII) for Early Detection of Improvement of Nutrition Status in Patients with Malnutrition

    OpenAIRE

    Suyama, Yohji; Adachi, Kyoichi; Notsu, Yoshitomo; Satoh, Emi; Nariai, Yukiko; Hashimoto, Yohko; Sumi, Asako; Kawaguchi, Mikiko; Ishimura, Norihisa

    2009-01-01

    Serum prealbumin level is useful for assessment of changes in nutritional status but it is markedly affected by the inflammation. In this study, we examined the efficacy of the corrected rapid turnover protein increment index (CRII) for prealbumin, which is calculated as [prealbumin level/C-reactive protein (CRP) level on the assessment day]/[prealbumin level/CRP level on the day of starting nutritional care], for prediction of improvement of nutritional status in patients with malnutrition. ...

  19. Casein and soya-bean protein have different effects on whole body protein turnover at the same nitrogen balance

    DEFF Research Database (Denmark)

    Nielsen, K; Kondrup, J; Elsner, Petteri

    1994-01-01

    , or hydrolysed soya-bean protein at a level of 9.1 g/kg BW per d. The diets, which were isoenergetic with the same carbohydrate: fat ratio, were given as a continuous intragastric infusion for at least 4 d. During the last 19 h 15N-glycine (a primed continuous infusion) was given intragastrically and 15N...... synthesis. The protein diets produced a positive N balance which was independent of the protein source. Intact and hydrolysed casein increased protein synthesis 2.6- and 2.0-fold respectively, compared with the protein-free diet. Protein degradation increased 1.4- and 1.2-fold respectively. Hydrolysed soya-bean...... protein did not increase protein synthesis but decreased protein degradation by 35% compared with the protein-free diet. Compared with the hydrolysed soya-bean protein, intact casein resulted in 2.2- and 2.8-fold higher rates of protein synthesis and degradation respectively. These results are not easily...

  20. Muscle protein turnover in rats treated with corticosterone (CC) or/and nandrolone decanoate (ND) and fed an adequate or a low-protein diet

    Energy Technology Data Exchange (ETDEWEB)

    Santidrian, S.; Cuevillas, F.; Goena, M.; Larralde, J.

    1986-03-01

    In order to investigate the possible antagonistic effect between glucocorticoids and androgens on muscle protein turnover, the authors have measured the fractional rates of gastrocnemius muscle protein synthesis (k/sub s/) and degradation (k/sub d/) by the constant-intravenous-infusion method using L-//sup 14/C/-tyrosine in rats receiving via s.c. per 100 g b.wt. 10 mg of CC, or 2 mg of ND or CC+ND at the indicated doses, and fed either an 18% or 5% protein diets over a period of 5 days. As an additional index of protein synthesis, RNA activity (g of synthesized protein/day/g RNA) was determined as well. Results showed that as compared to vehicle-injected animals fed the adequate diet, CC-treated rats exhibited a reduction of muscle k/sub d/, while ND-treated rats had an outstanding increase of muscle k/sub s/. However, rats receiving CC+ND showed k/sub s/ and k/sub d/ values similar to those displayed by control animals. Nevertheless, when the steroids were injected to rats fed the low-protein diet, CC has a catabolic effect on muscle protein but by reducing k/sub s/, while the anabolic action of ND is still displayed but by a significant reduction of muscle k/sub d/. CC+ND given to these protein-deficient rats caused an increase in muscle k/sub s/ and a reduction in k/sub d/. These results might indicate that, at least in part, ND antagonizes the catabolic action of high doses of CC on muscle protein metabolism.

  1. Muscle protein turnover in rats treated with corticosterone (CC) or/and nandrolone decanoate (ND) and fed an adequate or a low-protein diet

    International Nuclear Information System (INIS)

    Santidrian, S.; Cuevillas, F.; Goena, M.; Larralde, J.

    1986-01-01

    In order to investigate the possible antagonistic effect between glucocorticoids and androgens on muscle protein turnover, the authors have measured the fractional rates of gastrocnemius muscle protein synthesis (k/sub s/) and degradation (k/sub d/) by the constant-intravenous-infusion method using L-/ 14 C/-tyrosine in rats receiving via s.c. per 100 g b.wt. 10 mg of CC, or 2 mg of ND or CC+ND at the indicated doses, and fed either an 18% or 5% protein diets over a period of 5 days. As an additional index of protein synthesis, RNA activity (g of synthesized protein/day/g RNA) was determined as well. Results showed that as compared to vehicle-injected animals fed the adequate diet, CC-treated rats exhibited a reduction of muscle k/sub d/, while ND-treated rats had an outstanding increase of muscle k/sub s/. However, rats receiving CC+ND showed k/sub s/ and k/sub d/ values similar to those displayed by control animals. Nevertheless, when the steroids were injected to rats fed the low-protein diet, CC has a catabolic effect on muscle protein but by reducing k/sub s/, while the anabolic action of ND is still displayed but by a significant reduction of muscle k/sub d/. CC+ND given to these protein-deficient rats caused an increase in muscle k/sub s/ and a reduction in k/sub d/. These results might indicate that, at least in part, ND antagonizes the catabolic action of high doses of CC on muscle protein metabolism

  2. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

    DEFF Research Database (Denmark)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen

    2008-01-01

    , there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14)C content from the atmospheric carbon dioxide. The (14)C content of the lens proteins thus reflects the atmospheric content of (14)C when the lens crystallines were formed. Precise radiocarbon...

  3. Dystropathology increases energy expenditure and protein turnover in the Mdx mouse model of Duchenne muscular dystrophy

    Science.gov (United States)

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the diet...

  4. Amino acid metabolism and whole-body protein turnover in lambs ...

    African Journals Online (AJOL)

    Department of Microbiology, Biochemistry and Nutrition, University of New England, Armidale,. NSW 2351, Australia. The effect of protein supplementation of a wheat straw ... deur byvoeding met aminosure wat teen rumenafbraak beskerm is. I Extract from a PhD thesis submitted by PBC to the University of New England.

  5. Sphingomyelin synthases regulate protein trafficking and secretion.

    Directory of Open Access Journals (Sweden)

    Marimuthu Subathra

    Full Text Available Sphingomyelin synthases (SMS1 and 2 represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG. SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD, to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN, the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2 are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

  6. Distribution of sterol carrier protein2 (SCP2) in rat tissues and evidence for slow turnover in liver and adrenal cortex

    International Nuclear Information System (INIS)

    Kharroubi, A.; Chanderbhan, R.; Fiskum, G.; Noland, B.J.; Scallen, T.J.; Vahouny, G.V.

    1986-01-01

    Sterol carrier protein 2 (SCP 2 ) has been implicated in the regulation of the terminal stages of hepatic cholesterol biosynthesis, and in sterol utilization for adrenal steroid hormone and hepatic bile acid synthesis. In the present studies, a highly sensitive radioimmunoassay, using [ 125 I] SCP 2 , has been developed. Highest levels of SCP 2 were found in rat liver with progressively lower levels in intestinal mucosa, adrenal, kidney, lung and testis. SCP 2 levels were low or absent in heart, brain, skeletal muscle and serum. Liver SCP 2 was largely (44%) associated with the microsomal fraction, while in adrenal, 46% was associated with mitochondria, a distribution which is consistent with the proposed roles for SCP 2 in these tissues. Levels of SCP 2 in AS 30D hepatoma cells were only 5% of those in normal liver. In liver there was no indication of diurnal rhythm of SCP 2 in the cytosol and only slight variation of the microsomal SCP 2 levels. Fasting has only slight effects on SCP 2 concentration of rat liver microsomes and cytosol. Neither ACTH nor cycloheximide treatment of rats had a significant effect on SCP 2 distribution in the adrenal. In general, these findings indicate that SCP 2 has a low turn-over rate

  7. Effect of white striping myopathy on breast muscle (Pectoralis major) protein turnover and gene expression in broilers.

    Science.gov (United States)

    Vignale, Karen; Caldas, Justina V; England, Judy A; Boonsinchai, Nirun; Magnuson, Andrew; Pollock, Erik D; Dridi, Sami; Owens, Casey M; Coon, Craig N

    2017-04-01

    A study was conducted to evaluate the effect of white striping ( ) of broiler breast muscle ( Pectoralis major ) on protein turnover and gene expression of genes related to protein degradation and fatty acid synthesis. A total of 560 day-old male broiler chicks Cobb 500 were allocated in a total of 16 pens, 35 chicks per pen. A completely randomized design was conducted with a 2 × 3 factorial arrangement (2 scores: severe and normal, and 3 breast meat samples sites). At d 60, 20 birds were randomly selected, euthanized, and scored for white striping. Scoring was either normal ( , no WS) or severe ( ). Also, the same day, 17 birds (16 infused, one control) were randomly selected and infused with a solution of 15 N Phen 40% ( ). Breast muscle tissue was taken for gene expression analysis of the following genes: MuRF1, atrogin-1, IGF-1, insulin receptor ( ), fatty acid synthetase, and acetyl CoA carboxylase ( ). Each bird was humanely euthanized after 10 minutes of infusion and scored for WS (NORM or SEV). Samples of the breast muscle ( Pectoralis major ) were taken at different layers (3 samples per bird: ventral, medial, dorsal), along with a sample of excreta for 3-methylhistidine analysis. Out of the 16 breast samples taken, only 10 were selected for analysis based on the WS score (5 NORM and 5 SEV). No significant differences ( P > 0.05) were found in fractional synthesis rate ( ) between SEV WS, NORM and sample sites for breast meat. However, fractional breakdown rate ( ) was significantly higher in birds with SEV WS compared to NORM (8.2 and 4.28, respectively, P white striping are degrading more muscular protein and mobilizing more fat. © 2016 Poultry Science Association Inc.

  8. Whole-body protein turnover in malnourished patients with child class B and C cirrhosis on diets low to high in protein energy.

    Science.gov (United States)

    Dichi, J B; Dichi, I; Maio, R; Correa, C R; Angeleli, A Y; Bicudo, M H; Rezende, T A; Burini, R C

    2001-03-01

    The purpose of this study was to determine the rate of whole-body protein turnover in moderately and severely alcoholic, malnourished, cirrhotic patients fed with different amounts of protein or energy. Six male patients (Child classes B and C) and four age- and sex-matched healthy control subjects were studied for 18 d in fasting and feeding states; a single oral dose of [(15)N]glycine was used as a tracer and urinary ammonia was the end product. The kinetic study showed that patients had higher protein catabolism while fasting (patients: 3.14 +/- 1.2 g of lean body mass/9 h; controls: 1.8 +/- 0.3 g of lean body mass/9 h; P hyperproteic/hyperenergetic diet when compared with fasting. Nitrogen retention was consistent with the lower protein-catabolism rate; a statistically significant increase in nitrogen balance was observed when patients were fed with the hyperproteic/hyperenergetic diet compared with fasting (4.3 +/- 3.2 g of nitrogen/d and -2.2 +/- 1.9 g of nitrogen/d, respectively; P hyperproteic/hyperenergetic diet is likely needed to improve their clinical and nutritional status.

  9. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  10. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

    OpenAIRE

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A.; Salic, Adrian

    2011-01-01

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is s...

  11. Identifying molecular effects of diet through systems biology: influence of herring diet on sterol metabolism and protein turnover in mice.

    Directory of Open Access Journals (Sweden)

    Intawat Nookaew

    Full Text Available BACKGROUND: Changes in lifestyle have resulted in an epidemic development of obesity-related diseases that challenge the healthcare systems worldwide. To develop strategies to tackle this problem the focus is on diet to prevent the development of obesity-associated diseases such as cardiovascular disease (CVD. This will require methods for linking nutrient intake with specific metabolic processes in different tissues. METHODOLOGY/PRINCIPAL FINDING: Low-density lipoprotein receptor-deficient (Ldlr -/- mice were fed a high fat/high sugar diet to mimic a westernized diet, being a major reason for development of obesity and atherosclerosis. The diets were supplemented with either beef or herring, and matched in macronutrient contents. Body composition, plasma lipids and aortic lesion areas were measured. Transcriptomes of metabolically important tissues, e.g. liver, muscle and adipose tissue were analyzed by an integrated approach with metabolic networks to directly map the metabolic effects of diet in these different tissues. Our analysis revealed a reduction in sterol metabolism and protein turnover at the transcriptional level in herring-fed mice. CONCLUSION: This study shows that an integrated analysis of transcriptome data using metabolic networks resulted in the identification of signature pathways. This could not have been achieved using standard clustering methods. In particular, this systems biology analysis could enrich the information content of biomedical or nutritional data where subtle changes in several tissues together affects body metabolism or disease progression. This could be applied to improve diets for subjects exposed to health risks associated with obesity.

  12. Regulation of protein phosphorylation in oat mitochondria

    International Nuclear Information System (INIS)

    Pike, C.; Kopeck, K.; Sceppa, E.

    1989-01-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

  13. Processing and turnover of the Hedgehog protein in the endoplasmic reticulum.

    Science.gov (United States)

    Chen, Xin; Tukachinsky, Hanna; Huang, Chih-Hsiang; Jao, Cindy; Chu, Yue-Ru; Tang, Hsiang-Yun; Mueller, Britta; Schulman, Sol; Rapoport, Tom A; Salic, Adrian

    2011-03-07

    The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.

  14. Commitment Profiles and Employee Turnover

    Science.gov (United States)

    Stanley, Laura; Vandenberghe, Christian; Vandenberg, Robert; Bentein, Kathleen

    2013-01-01

    We examined how affective (AC), normative (NC), perceived sacrifice (PS), and few alternatives (FA) commitments combine to form profiles and determine turnover intention and turnover. We theorized that three mechanisms account for how profiles operate, i.e., the degree to which membership is internally regulated, the perceived desirability and…

  15. Specific DNA-binding proteins and DNA sequences involved in steroid hormone regulation of gene expression

    International Nuclear Information System (INIS)

    Spelsberg, T.; Hora, J.; Horton, M.; Goldberger, A.; Littlefield, B.; Seelke, R.; Toyoda, H.

    1987-01-01

    Steroid hormones circulate in the blood and are taken by target cells via complexes with intracellular binding proteins termed receptors, that are hormone and tissue specific. Each receptor binds it specific steroid with very high affinity, having an equilibrium dissociation constant (K/sub d/) in the range of 10 -9 to 10 -10 M. Once bound by their specific steroid hormones, the steroid receptors undergo a conformational change which allows them to bind with high affinity to sites on chromatin, termed nuclear acceptor sites. There are estimated 5,000 to 10,000 of these sites expressed with an equal number not expressed (''masked'') in intact chromatin. The result of the binding to nuclear acceptor sites is an alteration of gene transcription or, in some cases, gene expression as measured by the changing levels of specific RNAs and proteins in that target tissue. Each steroid regulates specific effects on the RNA and protein profiles. The chronology of the above mechanism of action after injection of radiolabelled steroid as is follows: Steroid-receptor complex formation (1 minute), nuclear acceptor sites (2 minutes), effects on RNA synthesis (10 to 30 minutes), and finally the changing protein profiles via changes in protein synthesis and protein turnover (1 to 6 hours). Thus steroid receptors represent one of the first identified intracellular gene regulation proteins. The receptor molecules themselves are regulated by the presence or absence of the steroid molecule

  16. ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis.

    Science.gov (United States)

    Jin, Shengfang; Chen, Jiang; Chen, Lizao; Histen, Gavin; Lin, Zhizhong; Gross, Stefan; Hixon, Jeffrey; Chen, Yue; Kung, Charles; Chen, Yiwei; Fu, Yufei; Lu, Yuxuan; Lin, Hui; Cai, Xiujun; Yang, Hua; Cairns, Rob A; Dorsch, Marion; Su, Shinsan M; Biller, Scott; Mak, Tak W; Cang, Yong

    2015-07-21

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.

  17. Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin.

    Directory of Open Access Journals (Sweden)

    Madhusudan Venkatareddy

    Full Text Available Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase, Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

  18. Protein trafficking and maturation regulate intramembrane proteolysis.

    Science.gov (United States)

    Morohashi, Yuichi; Tomita, Taisuke

    2013-12-01

    Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  20. Turnover Time

    Science.gov (United States)

    Ecosystems contain energy and materials such as carbon, nitrogen, phosphorus, and water, and are open to their flow-through. Turnover time refers to the amount of time required for replacement by flow-through of the energy or substance of interest contained in the system, and is ...

  1. Polycomb Group Protein PHF1 Regulates p53-dependent Cell Growth Arrest and Apoptosis*

    Science.gov (United States)

    Yang, Yang; Wang, Chenji; Zhang, Pingzhao; Gao, Kun; Wang, Dejie; Yu, Hongxiu; Zhang, Ting; Jiang, Sirui; Hexige, Saiyin; Hong, Zehui; Yasui, Akira; Liu, Jun O.; Huang, Haojie; Yu, Long

    2013-01-01

    Polycomb group protein PHF1 is well known as a component of a novel EED-EZH2·Polycomb repressive complex 2 complex and plays important roles in H3K27 methylation and Hox gene silencing. PHF1 is also involved in the response to DNA double-strand breaks in human cells, promotes nonhomologous end-joining processes through interaction with Ku70/Ku80. Here, we identified another function of PHF1 as a potential p53 pathway activator in a pathway screen using luminescence reporter assay. Subsequent studies showed PHF1 directly interacts with p53 proteins both in vivo and in vitro and co-localized in nucleus. PHF1 binds to the C-terminal regulatory domain of p53. Overexpression of PHF1 elevated p53 protein level and prolonged its turnover. Knockdown of PHF1 reduced p53 protein level and its target gene expression both in normal state and DNA damage response. Mechanically, PHF1 protects p53 proteins from MDM2-mediated ubiquitination and degradation. Furthermore, we showed that PHF1 regulates cell growth arrest and etoposide-induced apoptosis in a p53-dependent manner. Finally, PHF1 expression was significantly down-regulated in human breast cancer samples. Taken together, we establish PHF1 as a novel positive regulator of the p53 pathway. These data shed light on the potential roles of PHF1 in tumorigenesis and/or tumor progression. PMID:23150668

  2. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    Science.gov (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  3. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

    Science.gov (United States)

    Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

    2014-02-18

    Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

  4. A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

    Science.gov (United States)

    Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

    2017-12-08

    Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover

    DEFF Research Database (Denmark)

    Monaghan, Jacqueline; Matschi, Susanne; Shorinola, Oluwaseyi

    2014-01-01

    Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen...... the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28...

  6. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

    DEFF Research Database (Denmark)

    Hong, Xin; Nguyen, Thanh Hung; Chen, Qingfeng

    2014-01-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor...

  7. Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

    Science.gov (United States)

    Inomata, Yui; Nagasaka, Shouta; Miyate, Kazuki; Goto, Yuta; Hino, Chizuru; Toukairin, Chihiro; Higashio, Rieko; Ishida, Kinji; Saino, Tomoyuki; Hirose, Masamichi; Tsumura, Hideki; Sanbe, Atsushi

    2018-02-19

    Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  9. Alpha8 Integrin (Itga8 Signalling Attenuates Chronic Renal Interstitial Fibrosis by Reducing Fibroblast Activation, Not by Interfering with Regulation of Cell Turnover.

    Directory of Open Access Journals (Sweden)

    Ines Marek

    Full Text Available The α8 integrin (Itga8 chain contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells. In unilateral ureteral obstruction Itga8 is de novo expressed in the tubulointerstitium and a deficiency of Itga8 results in more severe renal fibrosis after unilateral ureteral obstruction. We hypothesized that the increased tubulointerstitial damage after unilateral ureteral obstruction observed in mice deficient for Itga8 is associated with altered tubulointerstitial cell turnover and apoptotic mechanisms resulting from the lack of Itga8 in cells of the tubulointerstitium. Induction of unilateral ureteral obstruction was achieved by ligation of the right ureter in mice lacking Itga8. Unilateral ureteral obstruction increased proliferation and apoptosis rates of tubuloepithelial and interstitial cells, however, no differences were observed in the tubulointerstitium of mice lacking Itga8 and wild type controls regarding fibroblast or proliferating cell numbers as well as markers of endoplasmic reticulum stress and apoptosis after unilateral ureteral obstruction. In contrast, unilateral ureteral obstruction in mice lacking Itga8 led to more pronounced tubulointerstitial cell activation i.e. to the appearance of more phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium. Furthermore, a more severe macrophage and T-cell infiltration was observed in these animals compared to controls. Thus, Itga8 seems to attenuate tubulointerstitial fibrosis in unilateral ureteral obstruction not via regulation of cell turnover, but via regulation of TGF-β signalling, fibroblast activation and/or immune cell infiltration.

  10. Spirits Trade After Reform in 1863: To the Issue of Efficiency of State Regulation of Spirits Turnover in Terms of Free Trade

    Directory of Open Access Journals (Sweden)

    Natalia Ye. Goryushkina

    2015-12-01

    Full Text Available On the grounds of published and non-published archive materials the article estimates efficiency of state regulation of spirits turnover in terms of excise taxation period. It also states types of spirits trade, types of public houses, groups of people that have obtained the right for wholesale, retail and that of small amount tradeoff spirits. License gaining procedures are widely described as well. The author pays significant attention to the areas in cities and countryside, where the public houses should not have been opened as well as the individuals who were admitted to spirits trade; also he states events which influenced the sale of spirits. The requirements to interior of public houses, working hours, quality and volumes of trade are also specified. It is pointed out that the officials of excise tax board were to control the regulations of spirit trade, their authority extended to public houses control in 1885. The author comes to the conclusion that regulatory means of excise tax was efficient and by increasing and decreasing of license cost, complication and simplification of new public houses opening all together allowed to regulate the turnover of spirits in terms of free trade thus influencing the number, types, density, range and quality of spirits. However, there was distinct ambiguity in Ministry of Finance policy during the stated excise taxation period. On the one hand it limited free sale of spirits, eradicating public houses which were considered detrimental. On the other hand, the Ministry was set not to notice abuse in trade which gave the opportunity to gain greater benefits at public cost from spirits sales.

  11. The Influence of Dietary Habits and Meat Consumption on Plasma 3‐Methylhistidine—A Potential Marker for Muscle Protein Turnover

    Science.gov (United States)

    Kochlik, Bastian; Gerbracht, Christiana; Grune, Tilman

    2018-01-01

    Scope 3‐Methylhistidine (3‐MH) as a potential biomarker for muscle protein turnover is influenced by meat intake but data on the impact of meat on plasma 3‐MH are scarce. We determined the association of plasma 3‐MH, 1‐methylhistidine (1‐MH), and creatinine with dietary habits and assessed the impact of a single white meat intervention during a meat‐free period. Methods and results Plasma 3‐MH, 1‐MH, and creatinine concentrations of healthy young omnivores (n = 19) and vegetarians (n = 16) were analyzed together with data on anthropometry, body composition, grip strength, and nutrition. After baseline measurements omnivores adhered to a meat‐free diet for 6 days and received a defined administration of chicken breast on day four. At baseline, omnivores had higher plasma 3‐MH and 1‐MH concentrations than vegetarians. White meat administration led to a slight increase in plasma 3‐MH in omnivores. The elevated 3‐MH concentrations significantly declined within 24 h after white meat intake. Conclusion 1‐MH concentrations in plasma seem to be suitable to display (white) meat consumption and its influence on 3‐MH plasma concentration. 3‐MH in plasma may be used as a biomarker for muscle protein turnover if subjects have not consumed meat in the previous 24 h. PMID:29573154

  12. Protein turnover and cellular stress in mildly and severely affected muscles from patients with limb girdle muscular dystrophy type 2I.

    Directory of Open Access Journals (Sweden)

    Simon Hauerslev

    Full Text Available Patients with Limb girdle muscular dystrophy type 2I (LGMD2I are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4 and healthy subjects (n = 4. The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC and neural cell adhesion molecule (NCAM and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

  13. Extracellular Redox Regulation of Intracellular Reactive Oxygen Generation, Mitochondrial Function and Lipid Turnover in Cultured Human Adipocytes.

    Directory of Open Access Journals (Sweden)

    Albert R Jones

    Full Text Available Many tissues play an important role in metabolic homeostasis and the development of diabetes and obesity. We hypothesized that the circulating redox metabolome is a master metabolic regulatory system that impacts all organs and modulates reactive oxygen species (ROS production, lipid peroxidation, energy production and changes in lipid turnover in many cells including adipocytes.Differentiated human preadipocytes were exposed to the redox couples, lactate (L and pyruvate (P, β-hydroxybutyrate (βOHB and acetoacetate (Acoc, and the thiol-disulfides cysteine/ cystine (Cys/CySS and GSH/GSSG for 1.5-4 hours. ROS measurements were done with CM-H2DCFDA. Lipid peroxidation (LPO was assessed by a modification of the thiobarbituric acid method. Lipolysis was measured as glycerol release. Lipid synthesis was measured as 14C-glucose incorporated into lipid. Respiration was assessed using the SeaHorse XF24 analyzer and the proton leak was determined from the difference in respiration with oligomycin and antimycin A.Metabolites with increasing oxidation potentials (GSSG, CySS, Acoc increased adipocyte ROS. In contrast, P caused a decrease in ROS compared with L. Acoc also induced a significant increase in both LPO and lipid synthesis. L and Acoc increased lipolysis. βOHB increased respiration, mainly due to an increased proton leak. GSSG, when present throughout 14 days of differentiation significantly increased fat accumulation, but not when added later.We demonstrated that in human adipocytes changes in the external redox state impacted ROS production, LPO, energy efficiency, lipid handling, and differentiation. A more oxidized state generally led to increased ROS, LPO and lipid turnover and more reduction led to increased respiration and a proton leak. However, not all of the redox couples were the same suggesting compartmentalization. These data are consistent with the concept of the circulating redox metabolome as a master metabolic regulatory system.

  14. S100 Proteins As an Important Regulator of Macrophage Inflammation

    Directory of Open Access Journals (Sweden)

    Chang Xia

    2018-01-01

    Full Text Available The S100 proteins, a family of calcium-binding cytosolic proteins, have a broad range of intracellular and extracellular functions through regulating calcium balance, cell apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation. The intracellular functions of S100 proteins involve interaction with intracellular receptors, membrane protein recruitment/transportation, transcriptional regulation and integrating with enzymes or nucleic acids, and DNA repair. The S100 proteins could also be released from the cytoplasm, induced by tissue/cell damage and cellular stress. The extracellular S100 proteins, serving as a danger signal, are crucial in regulating immune homeostasis, post-traumatic injury, and inflammation. Extracellular S100 proteins are also considered biomarkers for some specific diseases. In this review, we will discuss the multi-functional roles of S100 proteins, especially their potential roles associated with cell migration, differentiation, tissue repair, and inflammation.

  15. AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Wojtaszewski, Jørgen; Richter, Erik

    2009-01-01

    In skeletal muscle, the contraction-activated heterotrimeric 5'-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved...... in mitochondrial biogenesis and other aspects of promoting an oxidative muscle phenotype. Here, the current knowledge on the expression of AMPK subunits in human quadriceps muscle and evidence from rodent studies suggesting distinct AMPK subunit expression pattern in different muscle types is reviewed. Then......, the intensity and time dependence of AMPK activation in human quadriceps and rodent muscle are evaluated. Subsequently, a major part of this review critically examines the evidence supporting a necessary and/or sufficient role of AMPK in a broad spectrum of skeletal muscle contraction-relevant processes...

  16. Regulation of Cellular and Molecular Functions by Protein ...

    Indian Academy of Sciences (India)

    ... a high-energy linkage. The free energy of hydrolysis 1 of protein bound tyrosine phosphate ... protein kinases, cdc2 kinase (which regulates cell division cycle) and related cdc ... residues in response to extracellular signals such as hormones or growth factors. ... involved in regulating glycogen metabolism. The activity of.

  17. Regulation of Neuronal Protein Trafficking and Translocation by SUMOylation

    Directory of Open Access Journals (Sweden)

    Jeremy M. Henley

    2012-05-01

    Full Text Available Post-translational modifications of proteins are essential for cell function. Covalent modification by SUMO (small ubiquitin-like modifier plays a role in multiple cell processes, including transcriptional regulation, DNA damage repair, protein localization and trafficking. Factors affecting protein localization and trafficking are particularly crucial in neurons because of their polarization, morphological complexity and functional specialization. SUMOylation has emerged as a major mediator of intranuclear and nucleo-cytoplasmic translocations of proteins involved in critical pathways such as circadian rhythm, apoptosis and protein degradation. In addition, SUMO-regulated re-localization of extranuclear proteins is required to sustain neuronal excitability and synaptic transmission. Thus, SUMOylation is a key arbiter of neuronal viability and function. Here, we provide an overview of recent advances in our understanding of regulation of neuronal protein localization and translocation by SUMO and highlight exciting areas of ongoing research.

  18. Lrp4, a novel receptor for Dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth and turnover in vivo.

    Directory of Open Access Journals (Sweden)

    Hong Y Choi

    2009-11-01

    Full Text Available Lrp4 is a multifunctional member of the low density lipoprotein-receptor gene family and a modulator of extracellular cell signaling pathways in development. For example, Lrp4 binds Wise, a secreted Wnt modulator and BMP antagonist. Lrp4 shares structural elements within the extracellular ligand binding domain with Lrp5 and Lrp6, two established Wnt co-receptors with important roles in osteogenesis. Sclerostin is a potent osteocyte secreted inhibitor of bone formation that directly binds Lrp5 and Lrp6 and modulates both BMP and Wnt signaling. The anti-osteogenic effect of sclerostin is thought to be mediated mainly by inhibition of Wnt signaling through Lrp5/6 within osteoblasts. Dickkopf1 (Dkk1 is another potent soluble Wnt inhibitor that binds to Lrp5 and Lrp6, can displace Lrp5-bound sclerostin and is itself regulated by BMPs. In a recent genome-wide association study of bone mineral density a significant modifier locus was detected near the SOST gene at 17q21, which encodes sclerostin. In addition, nonsynonymous SNPs in the LRP4 gene were suggestively associated with bone mineral density. Here we show that Lrp4 is expressed in bone and cultured osteoblasts and binds Dkk1 and sclerostin in vitro. MicroCT analysis of Lrp4 deficient mutant mice revealed shortened total femur length, reduced cortical femoral perimeter, and reduced total femur bone mineral content (BMC and bone mineral density (BMD. Lumbar spine trabecular bone volume per total volume (BV/TV was significantly reduced in the mutants and the serum and urinary bone turnover markers alkaline phosphatase, osteocalcin and desoxypyridinoline were increased. We conclude that Lrp4 is a novel osteoblast expressed Dkk1 and sclerostin receptor with a physiological role in the regulation of bone growth and turnover, which is likely mediated through its function as an integrator of Wnt and BMP signaling pathways.

  19. Functional Dynamics within the Human Ribosome Regulate the Rate of Active Protein Synthesis.

    Science.gov (United States)

    Ferguson, Angelica; Wang, Leyi; Altman, Roger B; Terry, Daniel S; Juette, Manuel F; Burnett, Benjamin J; Alejo, Jose L; Dass, Randall A; Parks, Matthew M; Vincent, C Theresa; Blanchard, Scott C

    2015-11-05

    The regulation of protein synthesis contributes to gene expression in both normal physiology and disease, yet kinetic investigations of the human translation mechanism are currently lacking. Using single-molecule fluorescence imaging methods, we have quantified the nature and timing of structural processes in human ribosomes during single-turnover and processive translation reactions. These measurements reveal that functional complexes exhibit dynamic behaviors and thermodynamic stabilities distinct from those observed for bacterial systems. Structurally defined sub-states of pre- and post-translocation complexes were sensitive to specific inhibitors of the eukaryotic ribosome, demonstrating the utility of this platform to probe drug mechanism. The application of three-color single-molecule fluorescence resonance energy transfer (smFRET) methods further revealed a long-distance allosteric coupling between distal tRNA binding sites within ribosomes bearing three tRNAs, which contributed to the rate of processive translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Neurobeachin regulates neurotransmitter receptor trafficking to synapses

    NARCIS (Netherlands)

    Nair, R.; Lauks, J.; Jung, S; Cooke, N.E.; de Wit, H.; Brose, N.; Kilimann, M.W.; Verhage, M.; Rhee, J.

    2013-01-01

    The surface density of neurotransmitter receptors at synapses is a key determinant of synaptic efficacy. Synaptic receptor accumulation is regulated by the transport, postsynaptic anchoring, and turnover of receptors, involving multiple trafficking, sorting, motor, and scaffold proteins. We found

  1. Estimation of whole body protein turnover by L-[1-[sup 13]C]-leucine tracer experiment in post-viral hepatocirrhotic patients

    Energy Technology Data Exchange (ETDEWEB)

    Xinhua, Zhu; Zhenduo, Tang; Tengchang, Dai; Zongqin, Xia [Shanghai Second Medical Univ. (China)

    1993-08-01

    A constant infusion of L-[1-[sup 1]'3C]-leucine given to the subject and the plasma [sup 13]C-leucine enrichment and breath [sup 1]'3CO[sub 2] enrichment were measured with GC-MS and gas isotope ratio MS respectively. The plateau isotope enrichments reached were used to calculate the whole body protein synthesis and breakdown rates using a two pools model. 15 post-viral hepatocirrhotic patients and 6 normal adults were studied. The whole body protein synthesis and breakdown rates were both markedly increased in cirrhotic patients, with the breakdown rate higher than the synthesis rate and hence leading to a negative nitrogen balance. The changes of 7 decompensated cirrhotic patients were more severe than 8 compensated cirrhotic patients. The results suggest that the protein malnutrition and negative nitrogen balance of post-viral hepatocirrhotic patients are not simply due to low intake of protein and that the hyperactive turnover, especially the excessive breakdown of body protein might play an important role.

  2. Water turnover in reindeer. Some environmental implications

    International Nuclear Information System (INIS)

    Cameron, R.D.; Luick, J.R.; White, R.G.

    1982-01-01

    The nutritional and climatic effects on water turnover (determined using tritiated water) in reindeer are described. At low ambient temperatures (ca. -20 0 C) water turnover is related to protein and mineral intake, which is reflected in the relations of faecal and urinary water to nitrogen and ash excretion via these avenues. As ambient temperature increases, respiratory water, faecal water and urine flow increase, resulting in an increase in water turnover

  3. Ammonia treatment of wheat straw. 2. Efficiency of microbial protein synthesis, rumen microbial protein pool size and turnover, and small intestinal protein digestion in sheep.

    NARCIS (Netherlands)

    Oosting, S.J.; Viets, T.C.; Lammers-Wienhoven, S.C.W.; Bruchem, van J.

    1993-01-01

    Ammonia-treated wheat straw (AWS) was compared with untreated wheat straw (UWS) and untreated wheat straw supplemented with urea (SWS) in an experiment with 6 wether sheep. Microbial protein synthesis increased after ammonia treatment due to the higher intake of rumen degradable organic matter (OM).

  4. ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

    OpenAIRE

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

    2007-01-01

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

  5. Accelerated extracellular matrix turnover during exacerbations of COPD

    DEFF Research Database (Denmark)

    Sand, Jannie M B; Knox, Alan J; Lange, Peter

    2015-01-01

    progression. Extracellular matrix (ECM) turnover reflects activity in tissues and consequently assessment of ECM turnover may serve as biomarkers of disease activity. We hypothesized that the turnover of lung ECM proteins were altered during exacerbations of COPD. METHODS: 69 patients with COPD hospitalised...... of circulating fragments of structural proteins, which may serve as markers of disease activity. This suggests that patients with COPD have accelerated ECM turnover during exacerbations which may be related to disease progression....

  6. ASIC proteins regulate smooth muscle cell migration.

    Science.gov (United States)

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

    2008-03-01

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

  7. Tracer kinetics: Modelling of tracer behaviour in nonlinear and nonsteady state systems exemplified by the evaluation of protein turnover in plant organs

    International Nuclear Information System (INIS)

    Winkler, E.

    1991-01-01

    If nonlinear biological processes are investigated by means of tracer experiments they can be modelled with linear kinetic equations (compartment equations) as long as the total system is in a stationary state. But if nonstationary behaviour is included considerations on the kinetics of the individual processes are necessary. Within the range of biological and agricultural investigations especially first order reactions (constant fraction processes), zero order reactions (constant amount process) and saturation reactions (Michaelis-Menten-kinetics) are to be taken into account. A rigorous treatment of data based on system theory can be preceeded by graphic-algebraic procedure which may be more or less uncertain in its results but which can easily be handled. An example is given of methodological considerations concerning the combination of evaluation procedures and the discrimination between different reaction mechanisms. It treats protein turnover in 2 different parts of growing wheat plants investigated by means of an 15 N-tracer experiment. Whereas in a stationary system (upper stalk section) linear tracer equations were sufficient irrespective of the true reaction mechanism, for protein synthesis in the upper leaf as a nonstationary system it was necessary to decide between the hypotheses of a zero order and a first order reaction. In accordance with statements in the literature the unambiguous result was a combination of protein synthesis as a zero order process and of protein degradation as a first order process. (orig.) [de

  8. Protein Turnover and Cellular Stress in Mildly and Severely Affected Muscles from Patients with Limb Girdle Muscular Dystrophy Type 2I

    DEFF Research Database (Denmark)

    Hauerslev, Simon; Sveen, Marie-Louise; Vissing, John

    2013-01-01

    Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal...... by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal...... highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy....

  9. Turnover among healthcare professionals.

    Science.gov (United States)

    Wood, Ben D

    2009-01-01

    Turnover among healthcare professionals is a costly consequence. The existing body of knowledge on healthcare professional turnover is correlated with job satisfaction levels. A landmark study differentiated 2 areas of job satisfaction categories: satisfiers and dissatisfiers (intrinsic and extrinsic motivators). The aim of this article is to examine existing research on precursors of turnover, such as burnout behaviors experienced by healthcare professionals, job satisfaction levels, employee organizational commitment, health complications which precede turnover, some current strategies to reduce turnover, and some effects CEO turnover has on employee turnover intentions.

  10. Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-β1 in ovariectomized rats.

    Science.gov (United States)

    Li, S; Niu, G; Wu, Y; Du, G; Huang, C; Yin, X; Liu, Z; Song, C; Leng, H

    2016-02-01

    To explore the effect of vitamin D on turnover of articular cartilage with ovariectomy (OVX) induced OA, and to investigate transforming growth factor-β1 (TGF-β1) as a possible underlying mechanism mediated by 1α,25(OH)2D3. Sixty-six rats were randomly allocated into seven groups: sham plus control diet (SHAM+CTL), OVX+CTL diet, sham plus vitamin D-deficient (VDD) diet, OVX+VDD diet, and three groups of ovariectomized rats treated with different doses of 1α,25(OH)2D3. The cartilage erosion and the levels of serum 17β-estradiol, 1α,25(OH)2D3 and C-telopeptide of type II collagen (CTX-II) were measured. TGF-β1, type II Collagen (CII), matrix metalloproteinases (MMP)-9,-13 in articular cartilage were assessed by immunohistochemistry. TGF-β1 and CTX-II expression were measured in articular cartilage chondrocytes treated with/without tumor necrosis factor (TNF-α), 1α,25(OH)2D3, and TGF-β receptor inhibitor (SB505124) in vitro. Cartilage erosion due to OVX was significantly reduced in a dose-dependent manner by 1α,25(OH)2D3 supplementation, and exacerbated by VDD. The expressions of TGF-β1 and CII in articular cartilage were suppressed by OVX and VDD, and rescued by 1α,25(OH)2D3 supplementation. The expression of MMP-9,-13 in articular cartilage increased with OVX and VDD, and decreased with 1α,25(OH)2D3 supplementation. In vitro experiments showed that 1α,25(OH)2D3 increased the TGF-β1 expression of TNF-α stimulated chondrocytes in a dose-dependent manner. 1α,25(OH)2D3 significantly counteracted the increased CTX-II release due to TNF-α stimulation, and this effect was significantly suppressed by SB505124. VDD aggravated cartilage erosion, and 1α,25(OH)2D3 supplementation showed protective effects in OVX-induced OA partly through the TGF-β1 pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  11. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  12. Claudins, dietary milk proteins, and intestinal barrier regulation.

    Science.gov (United States)

    Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

    2013-01-01

    The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. © 2013 International Life Sciences Institute.

  13. Piezo proteins: regulators of mechanosensation and other cellular processes.

    Science.gov (United States)

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    Science.gov (United States)

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature. PMID:25305018

  15. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  16. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  17. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  18. Global turnover of histone post-translational modifications and variants in human cells

    Directory of Open Access Journals (Sweden)

    Zee Barry M

    2010-12-01

    Full Text Available Abstract Background Post-translational modifications (PTMs on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement. Results In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same

  19. Standardized 15N tracer methods for the evaluation of the plasma protein turnover in clinical practice. 1

    International Nuclear Information System (INIS)

    Bornhak, H.

    1984-01-01

    Methods for quantitative isolation of plasma proteins or groups of proteins (total plasma or serum proteins, fibrin, total globulines, α, β, γ-globolines, albumin) are described based on combination of chromatography with precipitation and extraction techniques. These methods are adapted to the special requirements of 15 N analysis. They can be performed in clinic-chemical standard laboratories without special apparatuses or devices. The described procedures are the biochemico-analytical basis for the quantitative evaluation of tracer kinetics data by means of mathematic modelling. (author)

  20. Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

    Science.gov (United States)

    Calder, Michele D; Watson, Patricia H; Watson, Andrew J

    2011-11-01

    During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

  1. Regulation of protein translation initiation in response to ionizing radiation

    International Nuclear Information System (INIS)

    Trivigno, Donatella; Bornes, Laura; Huber, Stephan M; Rudner, Justine

    2013-01-01

    Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR) blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells

  2. Regulation of protein translation initiation in response to ionizing radiation

    Directory of Open Access Journals (Sweden)

    Trivigno Donatella

    2013-02-01

    Full Text Available Abstract Background Proliferating tumor cells require continuous protein synthesis. De novo synthesis of most proteins is regulated through cap-dependent translation. Cellular stress such as ionizing radiation (IR blocks cap-dependent translation resulting in shut-down of global protein translation which saves resources and energy needed for the stress response. At the same time, levels of proteins required for stress response are maintained or even increased. The study aimed to analyze the regulation of signaling pathways controlling protein translation in response to IR and the impact on Mcl-1, an anti-apoptotic and radioprotective protein, which levels rapidly decline upon IR. Methods Protein levels and processing were analyzed by Western blot. The assembly of the translational pre-initiation complex was examined by Immunoprecipitation and pull-down experiments with 7-methyl GTP agarose. To analyze IR-induced cell death, dissipation of the mitochondrial membrane potential and DNA fragmentation were determined by flow cytometry. Protein levels of the different initiation factors were down-regulated using RNA interference approach. Results IR induced caspase-dependent cleavage of the translational initiation factors eIF4G1, eIF3A, and eIF4B resulting in disassembly of the cap-dependent initiation complex. In addition, DAP5-dependent initiation complex that regulates IRES-dependent translation was disassembled in response to IR. Moreover, IR resulted in dephosphorylation of 4EBP1, an inhibitor of cap-dependent translation upstream of caspase activation. However, knock-down of eIF4G1, eIF4B, DAP5, or 4EBP1 did not affect IR-induced decline of the anti-apoptotic protein Mcl-1. Conclusion Our data shows that cap-dependent translation is regulated at several levels in response to IR. However, the experiments indicate that IR-induced Mcl-1 decline is not a consequence of translational inhibition in Jurkat cells.

  3. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    Science.gov (United States)

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  4. Effect of plaster cast immobilization on the turnover rates of soluble proteins and lactate dehydrogenase isoenzymes of rabbit M. soleus

    Energy Technology Data Exchange (ETDEWEB)

    Edes, I.; Dosa, E.; Sohar, I.; Guba, F. (Orvostudomanyi Egyetem, Szeged (Hungary). Biokemiai Tanszek)

    1982-01-01

    In atrophized muscle the decreases of the activity of LDH isoenzymes can be explained partly by a 15 per cent decrease of the enzyme synthesis and partly by a 25 per cent increase in catabolism. The quantities of the soluble proteins and LDH were measured after intravenously administered /sup 3/H-leucin incorporation, from the musculus soleus. LDH was isolated by means of affinity chromatography. Radioactivity was determined in a Packard Tri-Carb scintillation counter. The synthesis rate of soluble proteins barely changed during immobilization. In the atrophized muscle the decrease of the amount of soluble proteins could be almost exclusively interpreted in terms of a 25 per cent enchancement of degradative process. The accelerated catabolism is most probably due to the proteolytic enzymes activated by immobilization.

  5. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    OpenAIRE

    Bagriantsev, Sviatoslav N.; Gracheva, Elena O.; Gallagher, Patrick G.

    2014-01-01

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular deve...

  6. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

  7. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...... amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P 

  8. Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise

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    Renae Jane Stefanetti

    2014-01-01

    Full Text Available Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP. Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE. We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60-75 years versus 10 younger (18-30 years healthy male subjects at basal as well as 2 hours after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased in older subjects. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signalling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown measurements following RE in older versus younger subjects.

  9. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  10. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

    2010-01-01

    -synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live...

  11. Control of striatal signaling by G protein regulators

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    Keqiang eXie

    2011-08-01

    Full Text Available Signaling via heterotrimeric G proteins plays a crucial role in modulating the responses of striatal neurons that ultimately shape core behaviors mediated by the basal ganglia circuitry, such as reward valuation, habit formation and movement coordination. Activation of G-protein-coupled receptors (GPCRs by extracellular signals activates heterotrimeric G proteins by promoting the binding of GTP to their α subunits. G proteins exert their effects by influencing the activity of key effector proteins in this region, including ion channels, second messenger enzymes and protein kinases. Striatal neurons express a staggering number of GPCRs whose activation results in the engagement of downstream signaling pathways and cellular responses with unique profiles but common molecular mechanisms. Studies over the last decade have revealed that the extent and duration of GPCR signaling are controlled by a conserved protein family named Regulator of G protein Signaling (RGS. RGS proteins accelerate GTP hydrolysis by the α subunits of G proteins, thus promoting deactivation of GPCR signaling. In this review, we discuss the progress made in understanding the roles of RGS proteins in controlling striatal G protein signaling and providing integration and selectivity of signal transmission. We review evidence on the formation of a macromolecular complex between RGS proteins and other components of striatal signaling pathways, their molecular regulatory mechanisms and impacts on GPCR signaling in the striatum obtained from biochemical studies and experiments involving genetic mouse models. Special emphasis is placed on RGS9-2, a member of the RGS family that is highly enriched in the striatum and plays critical roles in drug addiction and motor control.

  12. Lipid droplet meets a mitochondrial protein to regulate adipocyte lipolysis

    Science.gov (United States)

    In response to adrenergic stimulation, adipocytes undergo protein kinase A (PKA)-stimulated lipolysis. A key PKA target in this context is perilipin 1, a major regulator of lipolysis on lipid droplets (LDs). A study published in this issue of The EMBO Journal (Pidoux et al, 2011) identifies optic at...

  13. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

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    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  14. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

    Science.gov (United States)

    Asenjo, Ana; Villanueva, Nieves

    2016-01-04

    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

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    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  16. Heat Shock Proteins in Tendinopathy: Novel Molecular Regulators

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    Neal L. Millar

    2012-01-01

    Full Text Available Tendon disorders—tendinopathies—are the primary reason for musculoskeletal consultation in primary care and account for up to 30% of rheumatological consultations. Whilst the molecular pathophysiology of tendinopathy remains difficult to interpret the disease process involving repetitive stress, and cellular load provides important mechanistic insight into the area of heat shock proteins which spans many disease processes in the autoimmune community. Heat shock proteins, also called damage-associated molecular patterns (DAMPs, are rapidly released following nonprogrammed cell death, are key effectors of the innate immune system, and critically restore homeostasis by promoting the reconstruction of the effected tissue. Our investigations have highlighted a key role for HSPs in tendion disease which may ultimately affect tissue rescue mechanisms in tendon pathology. This paper aims to provide an overview of the biology of heat shock proteins in soft tissue and how these mediators may be important regulators of inflammatory mediators and matrix regulation in tendinopathy.

  17. Regulation of neuronal communication by G protein-coupled receptors.

    Science.gov (United States)

    Huang, Yunhong; Thathiah, Amantha

    2015-06-22

    Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Regulated eukaryotic DNA replication origin firing with purified proteins.

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    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

  19. Regulation of bone morphogenetic proteins in early embryonic development

    Science.gov (United States)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  20. Thioredoxin 1 regulation of protein S-desulfhydration

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    Youngjun Ju

    2016-03-01

    Full Text Available The importance of H2S in biology and medicine has been widely recognized in recent years, and protein S-sulfhydration is proposed to mediate the direct actions of H2S bioactivity in the body. Thioredoxin 1 (Trx1 is an important reducing enzyme that cleaves disulfides in proteins and acts as an S-denitrosylase. The regulation of Trx1 on protein S-sulfhydration is unclear. Here we showed that Trx1 facilitates protein S-desulfhydration. Overexpression of Trx1 attenuated the basal level and H2S-induced protein S-sulfhydration by direct interaction with S-sulfhydrated proteins, i.e., glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase. In contrast, knockdown of Trx1 mRNA expression by short interfering RNA or blockage of Trx1 redox activity with PX12 or 2,4-dinitrochlorobenzene enhanced protein S-sulfhydration. Mutation of cysteine-32 but not cysteine-35 in the Trp–Cys32–Gly–Pro–Cys35 motif eliminated the binding of Trx1 with S-sulfhydrated proteins and abolished the S-desulfhydrating effect of Trx1. All these data suggest that Trx1 acts as an S-desulfhydrase.

  1. Identification of proteins regulated by curcumin in cerebral ischemia.

    Science.gov (United States)

    Shah, Fawad-Ali; Gim, Sang-Ah; Sung, Jin-Hee; Jeon, Seong-Jun; Kim, Myeong-Ok; Koh, Phil-Ok

    2016-03-01

    Curcumin is known to have a neuroprotective effect against cerebral ischemia. The objective of this study was to identify various proteins that are differentially expressed by curcumin treatment in focal cerebral ischemia using a proteomic approach. Adult male rats were treated with vehicle or curcumin 1 h after middle cerebral artery occlusion. Brain tissues were collected 24 h after the onset of middle cerebral artery occlusion, and cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. We detected several proteins with altered expression levels between vehicle- and curcumin-treated animals. Among these proteins, ubiquitin carboxy-terminal hydrolase L1, isocitrate dehydrogenase, adenosylhomocysteinase, and eukaryotic initiation factor 4A were decreased in the vehicle-treated animal, and curcumin treatment attenuated the injury-induced decreases of these proteins. Conversely, pyridoxal phosphate phosphatase was increased in the vehicle-treated animal, and curcumin treatment prevented decreases in this protein. The identified altered proteins are associated with cellular metabolism and differentiation. The results of this study suggest that curcumin exerts a neuroprotective effect by regulating the expression of various proteins in focal cerebral ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  3. Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

    Science.gov (United States)

    Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

    2012-01-01

    Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

  4. Acute myotube protein synthesis regulation by IL-6-related cytokines.

    Science.gov (United States)

    Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

    2017-11-01

    IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the

  5. Monitoring protein turnover during phosphate starvation-dependent autophagic degradation using a photoconvertible fluorescent protein aggregate in tobacco BY-2 cells.

    Science.gov (United States)

    Tasaki, Maiko; Asatsuma, Satoru; Matsuoka, Ken

    2014-01-01

    We have developed a system for quantitative monitoring of autophagic degradation in transformed tobacco BY-2 cells using an aggregate-prone protein comprised of cytochrome b5 (Cyt b5) and a tetrameric red fluorescent protein (RFP). Unfortunately, this system is of limited use for monitoring the kinetics of autophagic degradation because the proteins synthesized before and after induction of autophagy cannot be distinguished. To overcome this problem, we developed a system using kikume green-red (KikGR), a photoconvertible and tetrameric fluorescent protein that changes its fluorescence from green to red upon irradiation with purple light. Using the fusion protein of Cyt b5 and KikGR together with a method for the bulk conversion of KikGR, which we had previously used to convert the Golgi-localized monomeric KikGR fusion protein, we were able to monitor both the growth and de novo formation of aggregates. Using this system, we found that tobacco cells do not cease protein synthesis under conditions of phosphate (Pi)-starvation. Induction of autophagy under Pi-starvation, but not under sugar- or nitrogen-starvation, was specifically inhibited by phosphite, which is an analog of Pi with a different oxidation number. Therefore, the mechanism by which BY-2 cells can sense Pi-starvation and induce autophagy does not involve sensing a general decrease in energy supply and a specific Pi sensor might be involved in the induction of autophagy under Pi-starvation.

  6. Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

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    Jocelyn Widagdo

    2017-10-01

    Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

  7. Wrecked regulation of intrinsically disordered proteins in diseases: Pathogenicity of deregulated regulators

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    Vladimir N. Uversky

    2014-07-01

    Full Text Available Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs are typically related to regulation, signaling and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases.

  8. Time-dependent, glucose-regulated Arabidopsis Regulator of G-protein Signaling 1 network

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    Dinesh Kumar Jaiswal

    2016-04-01

    Full Text Available Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS. A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119 were not known previously. AtRGS1 protein interactions were dynamically modulated by d-glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

  9. Platelet activation by bacterial phospholipase C involves phosphoinositide turnover and phosphorylation of 47,000 dalton but not 20,000 dalton protein

    International Nuclear Information System (INIS)

    Huzoor-Akbar; Anwer, K.

    1986-01-01

    This study was conducted to examine the role of phosphoinositides (PIns) and phosphorylation of 47,000 dalton (P47) and 20,000 dalton (P20) proteins in platelet activation by bacterial phospholipase C (PLC). PLC induced serotonin secretion (SS) and platelet aggregation (PA) in a concentration dependent manner. PLC (0.02 U/ml) caused phosphorylation of P47 in a time dependent manner (27% at 0.5 min to 378% at 7 min). PLC did not induce more than 15% phosphorylation of P20 by 7 min. Aspirin (500 μM) blocked phosphorylation of P20 but did not inhibit SS, PA or phosphorylation of P47. PLC (0.04 U/ml) decreased radioactivity (cpm) in 32 P labeled phosphatidylinositol (PI), PI-4,5-bis-PO4 (PIP2) and PI-4-PO4 (PIP) by 20%, 12% and 7.5% respectively at 15 sec. The level of PI but not that of PIP2 returned to base line in 3 min. PIP level increased above control values within one min. PLC increased phosphatidic acid level (75% at 0.5 min. to 1545% at 3 min). In other experiments PLC produced diacylglycerol (DAG) in a time and concentration dependent manner. However, no DAG was detectable in the first 60 sec. These data suggest that: (a) PIns turnover and phosphorylation of P47 but not that of P20 is involved in platelet activation by PLC; and (b) DAG production from outer membrane phospholipids is not a prerequisite for platelet activation by PLC

  10. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  11. Chromatin-regulating proteins as targets for cancer therapy

    International Nuclear Information System (INIS)

    Oike, Takahiro; Ogiwara, Hideaki; Kohno, Takashi; Amornwichet, Napapat; Nakano, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. (author)

  12. Relationship between Protein Accumulation Regulation and Yield Formation in Soybean

    Institute of Scientific and Technical Information of China (English)

    CHEN Lihua; LI Jie; LIU Lijun; ZU Wei

    2006-01-01

    Three different genotypes soybeans were adopted in this experiment under three fertilizer levels.The object of this study was to investigate protein accumulation regulation of soybean cultivars under the condition of different nutrient levels, and their effects on soybean yield and quality, and to provide theoretical evidence for breed, cultivation and agricultural production, also man-powered controllable locations. The concentration of N in the leaves declined after seedling stage, then increased again at stage of early flowering, and started to decrease up to leaf senescence, declined rapidly from seed-filling season to stage of yellow ripeness. The concentration of N in the stems and pod walls declined with growth stage. High seed protein genotypes exhibited higher N assimilating and partitioning during whole growth stages. Pod walls were media of N partitioning. Protein was accumulated mainly during the later period of reproductive growth stage up to harvest, so plant growth after stage of yellow ripeness could not be neglected.

  13. Heat Shock Protein 90 regulates encystation in Entamoeba

    Directory of Open Access Journals (Sweden)

    Meetali eSingh

    2015-10-01

    Full Text Available Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90 in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

  14. Myostatin-like proteins regulate synaptic function and neuronal morphology.

    Science.gov (United States)

    Augustin, Hrvoje; McGourty, Kieran; Steinert, Joern R; Cochemé, Helena M; Adcott, Jennifer; Cabecinha, Melissa; Vincent, Alec; Halff, Els F; Kittler, Josef T; Boucrot, Emmanuel; Partridge, Linda

    2017-07-01

    Growth factors of the TGFβ superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth. © 2017. Published by The Company of Biologists Ltd.

  15. Central regulation of metabolism by protein tyrosine phosphatases

    Directory of Open Access Journals (Sweden)

    Ryan eTsou

    2013-01-01

    Full Text Available Protein tyrosine phosphatases (PTPs are important regulators of intracellular signaling pathways via the dephosphorylation of phosphotyrosyl residues on various receptor and non-receptor substrates. The phosphorylation state of central nervous system (CNS signaling components underlies the molecular mechanisms of a variety of physiological functions including the control of energy balance and glucose homeostasis. In this review, we summarize the current evidence implicating PTPs as central regulators of metabolism, specifically highlighting their interactions with the neuronal leptin and insulin signaling pathways. We discuss the role of a number of PTPs (PTP1B, SHP2, TCPTP, RPTPe, and PTEN, reviewing the findings from genetic mouse models and in vitro studies which highlight these phosphatases as key central regulators of energy homeostasis.

  16. Protein-protein interactions in the regulation of WRKY transcription factors.

    Science.gov (United States)

    Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-03-01

    It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

  17. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  18. Regulation of protein synthesis during sea urchin early development

    International Nuclear Information System (INIS)

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

  19. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    Science.gov (United States)

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  20. EML proteins in microtubule regulation and human disease.

    Science.gov (United States)

    Fry, Andrew M; O'Regan, Laura; Montgomery, Jessica; Adib, Rozita; Bayliss, Richard

    2016-10-15

    The EMLs are a conserved family of microtubule-associated proteins (MAPs). The founding member was discovered in sea urchins as a 77-kDa polypeptide that co-purified with microtubules. This protein, termed EMAP for echinoderm MAP, was the major non-tubulin component present in purified microtubule preparations made from unfertilized sea urchin eggs [J. Cell Sci. (1993) 104: , 445-450; J. Cell Sci. (1987) 87: (Pt 1), 71-84]. Orthologues of EMAP were subsequently identified in other echinoderms, such as starfish and sand dollar, and then in more distant eukaryotes, including flies, worms and vertebrates, where the name of ELP or EML (both for EMAP-like protein) has been adopted [BMC Dev. Biol. (2008) 8: , 110; Dev. Genes Evol. (2000) 210: , 2-10]. The common property of these proteins is their ability to decorate microtubules. However, whether they are associated with particular microtubule populations or exercise specific functions in different microtubule-dependent processes remains unknown. Furthermore, although there is limited evidence that they regulate microtubule dynamics, the biochemical mechanisms of their molecular activity have yet to be explored. Nevertheless, interest in these proteins has grown substantially because of the identification of EML mutations in neuronal disorders and oncogenic fusions in human cancers. Here, we summarize our current knowledge of the expression, localization and structure of what is proving to be an interesting and important class of MAPs. We also speculate about their function in microtubule regulation and highlight how the studies of EMLs in human diseases may open up novel avenues for patient therapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  1. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...... and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...

  2. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Su, Xiong [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Liu, Jialiu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Sundaresan, Sinju [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Stahl, Philip D., E-mail: pstahl@wustl.edu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  3. DMPD: Post-transcriptional regulation of proinflammatory proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15075353 Post-transcriptional regulation of proinflammatory proteins. Anderson P, P...l) (.csml) Show Post-transcriptional regulation of proinflammatory proteins. PubmedID 15075353 Title Post-tr...anscriptional regulation of proinflammatory proteins. Authors Anderson P, Phillip

  4. Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

    Directory of Open Access Journals (Sweden)

    Eduardo Cebollero

    2012-01-01

    Full Text Available During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

  5. Fat-specific protein 27 regulates storage of triacylglycerol

    DEFF Research Database (Denmark)

    Keller, P.; Petrie, J.T.; Rose, P. De

    2008-01-01

    FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins...... in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L...... decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement Udgivelsesdato: 2008/5/23...

  6. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate...... MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact...

  7. Regulation of Lipid and Glucose Metabolism by Phosphatidylcholine Transfer Protein

    Science.gov (United States)

    Kang, Hye Won; Wei, Jie; Cohen, David E.

    2010-01-01

    Phosphatidylcholine transfer protein (PC-TP, a.k.a. StARD2) binds phosphatidylcholines and catalyzes their intermembrane transfer and exchange in vitro. The structure of PC-TP comprises a hydrophobic pocket and a well-defined head-group binding site, and its gene expression is regulated by peroxisome proliferator activated receptor α. Recent studies have revealed key regulatory roles for PC-TP in lipid and glucose metabolism. Notably, Pctp−/− mice are sensitized to insulin action and exhibit more efficient brown fat-mediated thermogenesis. PC-TP appears to limit access of fatty acids to mitochondria by stimulating the activity of thioesterase superfamily member 2, a newly characterized long-chain fatty acyl-CoA thioesterase. Because PC-TP discriminates among phosphatidylcholines within lipid bilayers, it may function as a sensor that links metabolic regulation to membrane composition. PMID:20338778

  8. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    Directory of Open Access Journals (Sweden)

    Niria Treviño-Saldaña

    2017-01-01

    Full Text Available Modulation of posttranslational modifications (PTMs, such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the development and progression of cardiac dysfunction. Several phytochemicals exert cardioprotective effects by regulating protein acetylation levels. These effects are mainly exerted via activation of Sirt1 and Sirt3 and inhibition of acetyltransferases. Numerous studies support a cardioprotective role for sirtuin activators (e.g., resveratrol, as well as other emerging modulators of protein acetylation, including curcumin, honokiol, oroxilyn A, quercetin, epigallocatechin-3-gallate, bakuchiol, tyrosol, and berberine. Studies also point to a cardioprotective role for various nonaromatic molecules, such as docosahexaenoic acid, alpha-lipoic acid, sulforaphane, and caffeic acid ethanolamide. Here, we review the vast evidence from the bench to the clinical setting for the potential cardioprotective roles of various phytochemicals in the modulation of sirtuin-mediated deacetylation.

  9. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    Science.gov (United States)

    2017-01-01

    Modulation of posttranslational modifications (PTMs), such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the development and progression of cardiac dysfunction. Several phytochemicals exert cardioprotective effects by regulating protein acetylation levels. These effects are mainly exerted via activation of Sirt1 and Sirt3 and inhibition of acetyltransferases. Numerous studies support a cardioprotective role for sirtuin activators (e.g., resveratrol), as well as other emerging modulators of protein acetylation, including curcumin, honokiol, oroxilyn A, quercetin, epigallocatechin-3-gallate, bakuchiol, tyrosol, and berberine. Studies also point to a cardioprotective role for various nonaromatic molecules, such as docosahexaenoic acid, alpha-lipoic acid, sulforaphane, and caffeic acid ethanolamide. Here, we review the vast evidence from the bench to the clinical setting for the potential cardioprotective roles of various phytochemicals in the modulation of sirtuin-mediated deacetylation. PMID:29234485

  10. The PTEN protein: cellular localization and post-translational regulation.

    Science.gov (United States)

    Leslie, Nick R; Kriplani, Nisha; Hermida, Miguel A; Alvarez-Garcia, Virginia; Wise, Helen M

    2016-02-01

    The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation. © 2016 Authors; published by Portland Press Limited.

  11. A Dictyostelium chalone uses G proteins to regulate proliferation

    Directory of Open Access Journals (Sweden)

    Hanson Nana E

    2009-07-01

    Full Text Available Abstract Background Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. Results We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA. Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus, whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. Conclusion This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  12. A Dictyostelium chalone uses G proteins to regulate proliferation.

    Science.gov (United States)

    Bakthavatsalam, Deenadayalan; Choe, Jonathan M; Hanson, Nana E; Gomer, Richard H

    2009-07-27

    Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  13. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    Science.gov (United States)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  14. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

    Directory of Open Access Journals (Sweden)

    Boyce Mark

    2012-08-01

    Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

  15. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

    Science.gov (United States)

    Boyce, Mark; Celma, Cristina C P; Roy, Polly

    2012-08-29

    Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

  16. Managing teacher turnover

    African Journals Online (AJOL)

    Erna Kinsey

    - ... vacant teaching posts are not filled (Pretorius & Heard, 1999; Anon., ... it reaches critical proportions. This article therefore investigates teacher turnover as a phenome- non to be managed and suggests ways of doing so. The study is based.

  17. Translational regulation of ribosomal protein S15 drives characteristic patterns of protein-mRNA epistasis.

    Science.gov (United States)

    Mallik, Saurav; Basu, Sudipto; Hait, Suman; Kundu, Sudip

    2018-04-21

    Do coding and regulatory segments of a gene co-evolve with each-other? Seeking answers to this question, here we analyze the case of Escherichia coli ribosomal protein S15, that represses its own translation by specifically binding its messenger RNA (rpsO mRNA) and stabilizing a pseudoknot structure at the upstream untranslated region, thus trapping the ribosome into an incomplete translation initiation complex. In the absence of S15, ribosomal protein S1 recognizes rpsO and promotes translation by melting this very pseudoknot. We employ a robust statistical method to detect signatures of positive epistasis between residue site pairs and find that biophysical constraints of translational regulation (S15-rpsO and S1-rpsO recognition, S15-mediated rpsO structural rearrangement, and S1-mediated melting) are strong predictors of positive epistasis. Transforming the epistatic pairs into a network, we find that signatures of two different, but interconnected regulatory cascades are imprinted in the sequence-space and can be captured in terms of two dense network modules that are sparsely connected to each other. This network topology further reflects a general principle of how functionally coupled components of biological networks are interconnected. These results depict a model case, where translational regulation drives characteristic residue-level epistasis-not only between a protein and its own mRNA but also between a protein and the mRNA of an entirely different protein. © 2018 Wiley Periodicals, Inc.

  18. Mitochondrial fission proteins regulate programmed cell death in yeast.

    Science.gov (United States)

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  19. Regulation of the autophagy protein LC3 by phosphorylation

    Science.gov (United States)

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  20. Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

    Directory of Open Access Journals (Sweden)

    Zhenyong Wu

    2017-10-01

    Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

  1. Curcumin as a natural regulator of monocyte chemoattractant protein-1.

    Science.gov (United States)

    Karimian, Maryam Saberi; Pirro, Matteo; Majeed, Muhammed; Sahebkar, Amirhossein

    2017-02-01

    Monocyte chemoattractant/chemotactic protein-1 (MCP-1), a member of the CC chemokine family, is one of the key chemokines that regulate migration and tissue infiltration of monocytes/macrophages. Its role in the pathophysiology of several inflammatory diseases has been widely recognized, thus making MCP-1 a possible target for anti-inflammatory treatments. Curcumin (diferuloylmethane) is a natural polyphenol derived from the rhizomes of Curcuma Longa L. (turmeric). Anti-inflammatory action underlies numerous pharmacological effects of curcumin in the control and prevention of several diseases. The purpose of this review is to evaluate the effects of curcumin on the regulation of MCP-1 as a key mediator of chemotaxis and inflammation, and the biological consequences thereof. In vitro studies have shown that curcumin can decrease MCP-1 production in various cell lines. Animal studies have also revealed that curcumin can attenuate MCP-1 expression and improve a range of inflammatory diseases through multiple molecular targets and mechanisms of action. There is limited data from human clinical trials showing the decreasing effect of curcumin on MCP-1 concentrations and improvement of the course of inflammatory diseases. Most of the in vitro and animal studies confirm that curcumin exert its MCP-1-lowering and anti-inflammatory effects by down-regulating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway. As yet, there is limited data from human clinical trials showing the effect of curcumin on MCP-1 levels and improvement of the course of inflammatory diseases. More evidence, especially from human studies, is needed to better assess the effects of curcumin on circulating MCP-1 in different human diseases and the role of this modulatory effect in the putative anti-inflammatory properties of curcumin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  3. Regulation of G Protein-Coupled Receptors by Ubiquitination

    Directory of Open Access Journals (Sweden)

    Kamila Skieterska

    2017-04-01

    Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

  4. Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

    Directory of Open Access Journals (Sweden)

    José Luis Carrasco

    Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

  5. Stress responses during ageing: molecular pathways regulating protein homeostasis.

    Science.gov (United States)

    Kyriakakis, Emmanouil; Princz, Andrea; Tavernarakis, Nektarios

    2015-01-01

    The ageing process is characterized by deterioration of physiological function accompanied by frailty and ageing-associated diseases. The most broadly and well-studied pathways influencing ageing are the insulin/insulin-like growth factor 1 signaling pathway and the dietary restriction pathway. Recent studies in diverse organisms have also delineated emerging pathways, which collectively or independently contribute to ageing. Among them the proteostatic-stress-response networks, inextricably affect normal ageing by maintaining or restoring protein homeostasis to preserve proper cellular and organismal function. In this chapter, we survey the involvement of heat stress and endoplasmic reticulum stress responses in the regulation of longevity, placing emphasis on the cross talk between different response mechanisms and their systemic effects. We further discuss novel insights relevant to the molecular pathways mediating these stress responses that may facilitate the development of innovative interventions targeting age-related pathologies such as diabetes, cancer, cardiovascular and neurodegenerative diseases.

  6. Shoc2/Sur8 protein regulates neurite outgrowth.

    Directory of Open Access Journals (Sweden)

    Gonzalo Leon

    Full Text Available The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.

  7. Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix

    DEFF Research Database (Denmark)

    Kehlet, Stephanie N.; Willumsen, Nicholas; Armbrecht, Gabriele

    2018-01-01

    The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small...... collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I...... an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies....

  8. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  9. A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

    Science.gov (United States)

    Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

    2001-12-15

    Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

  10. G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells*

    OpenAIRE

    Vellano, Christopher P.; Maher, Ellen M.; Hepler, John R.; Blumer, Joe B.

    2011-01-01

    Background: Regulator of G protein signaling 14 (RGS14) is a G protein regulatory (GPR) protein that participates in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs).

  11. Regulation and Selectivity of Exchange Factors for G-proteins of the Ras-family

    NARCIS (Netherlands)

    Popovic, M.

    2013-01-01

    Small G-proteins are important regulators of the cellular signaling pathways. Among them, members of the Ras family of small G-proteins regulate processes such as cell differentiation, growth, migration, transport and adhesion, and their deregulation may lead to various diseases. Small G-proteins

  12. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    Science.gov (United States)

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  13. Leucocyte protein Trojan, a possible regulator of apoptosis.

    Science.gov (United States)

    Petrov, Petar; Syrjänen, Riikka; Uchida, Tatsuya; Vainio, Olli

    2017-02-01

    Trojan is a leucocyte-specific protein, cloned from chicken embryonic thymocyte cDNA library. The molecule is a type I transmembrane protein with an extracellular CCP domain, followed by two FN3 domains. Its cytoplasmic tail is predicted to possess a MAPK docking and a PKA phosphorylation sites. Trojan has been proposed to have an anti-apoptotic role based on its differential expression on developing thymocyte subpopulations. Using a chicken cell line, our in vitro studies showed that upon apoptosis induction, Trojan expression rises dramatically on the surface of surviving cells and gradually decreases towards its normal levels as cells recover. When sorted based on their expression levels of Trojan, cells with high expression appeared less susceptible to apoptotic induction than those bearing no or low levels of Trojan on their surface. The mechanism by which the molecule exerts its function is yet to be discovered. We found that cells overexpressing Trojan from a cDNA plasmid show elevated steady-state levels of intracellular calcium, suggesting the molecule is able to transmit cytoplasmic signals. The mechanistic nature of Trojan-induced signalling is a target of future investigation. In this article, we conducted a series of experiments that suggest Trojan as an anti-apoptotic regulator. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  14. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Floss, Daniela S; Levy, Julien G; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J

    2013-12-17

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

  15. ANTESEDEN EMPLOYEE TURNOVER

    Directory of Open Access Journals (Sweden)

    Heryadi Fardilah

    2018-03-01

    Full Text Available This research is concerning the effect of workplace environment, job satisfaction, and  job performance on employee turnover, and the background is to get the convenience of employees condition, the satisfaction of job results, maximum performance, and keep and reduce in and out employees. The purpose of this research is to see how far the influence of workplace environment, job satisfaction, and job performance on employee turnover. Planning of this research uses primary data that is got by handing over questioners to 200 employees in Telecommunication company in East Jakarta. The analysis method which is used is double regresi.  Results of  this  research give conclusion that there's a positive and significant influence of workplace environment, job satisfaction, and job performance on employee turnover.

  16. Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

    Science.gov (United States)

    Scapa, Erez F; Pocai, Alessandro; Wu, Michele K; Gutierrez-Juarez, Roger; Glenz, Lauren; Kanno, Keishi; Li, Hua; Biddinger, Sudha; Jelicks, Linda A; Rossetti, Luciano; Cohen, David E

    2008-07-01

    Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

  17. A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study

    International Nuclear Information System (INIS)

    Corfe, Bernard M; Williams, Elizabeth A; Bury, Jonathan P; Riley, Stuart A; Croucher, Lisa J; Lai, Daphne YL; Evans, Caroline A

    2009-01-01

    A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. There are two principal arms to this study: (i) a cross-sectional study (FACT OBS) of 90 subjects recruited from gastroenterology clinics and; (ii) an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. Trial Registration Number: ISRCTN90852168

  18. A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study

    Directory of Open Access Journals (Sweden)

    Croucher Lisa J

    2009-09-01

    Full Text Available Abstract Background A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. Methods an design There are two principal arms to this study: (i a cross-sectional study (FACT OBS of 90 subjects recruited from gastroenterology clinics and; (ii an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. Discussion This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. Trial Registration Trial Registration Number: ISRCTN90852168

  19. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

    2014-01-01

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  20. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  1. Regulation of hematopoietic cell function by protein tyrosine kinase-encoding oncogenes, a review

    NARCIS (Netherlands)

    Punt, C. J.

    1992-01-01

    Tyrosine phosphorylation of proteins by protein tyrosine kinases (PTKs) is an important mechanism in the regulation of various cellular processes such as proliferation, differentiation, and transformation. Accumulating data implicate PTKs as essential intermediates in the transduction of

  2. Regulation of complement by cartilage oligomeric matrix protein allows for a novel molecular diagnostic principle in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Happonen, Kaisa E; Saxne, Tore; Aspberg, Anders

    2010-01-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthr...

  3. Costs of employee turnover

    Directory of Open Access Journals (Sweden)

    Jiří Duda

    2013-01-01

    Full Text Available The aim of this paper is to establish a general methodology for calculating the costs incurred by employee turnover. This paper deals with identification of costs incurred by the departure of an employee, and does not deal with the cost of recruitment of a new employee. Economic calculations are adjusted to the tax policy in the Czech Republic. The costs of employee turnover (according to Bliss, 2012 include the costs of substitution of the unoccupied position, costs of conducting the exit interview and termination of the contract. The cost of an executive’s time to understand the causes of leaving and costs of the leaving employee’s training were also determined. Important factors in the costs of employee turnover also include the loss of knowledge and possibly also a loss of customers. Costs of lost employee and department productiveness represent an important part of the costs of employee turnover, as well. For all of these costs there have been proposed general calculations formulas.

  4. Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

    OpenAIRE

    Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

    2008-01-01

    The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

  5. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  6. F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

    Science.gov (United States)

    Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

    2014-03-07

    The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

  7. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  8. The effects of calcium regulation of endosperm reserve protein ...

    African Journals Online (AJOL)

    The effects of steep liquor calcium ion on sorghum endosperm reserve protein mobilization were evaluated using two improved Nigeria sorghum cultivars (ICSV 400 and KSV 8). The key protein modification factors evaluated were free amino nitrogen (FAN), total non protein nitrogen (TNPN) and soluble protein of cold water ...

  9. Regulation of PCNA-protein interactions for genome stability

    DEFF Research Database (Denmark)

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  11. Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

    International Nuclear Information System (INIS)

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-01-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H 2 O 2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H 2 O 2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H 2 O 2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

  12. The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover

    DEFF Research Database (Denmark)

    Luo, Guangzuo; Gruhler, Albrecht; Liu, Ying

    2008-01-01

    Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1...... that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We...

  13. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

    Science.gov (United States)

    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

  14. Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

    DEFF Research Database (Denmark)

    Djidja, Marie-Claude; Chang, Joan; Hadjiprocopis, Andreas

    2014-01-01

    Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using...

  15. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  16. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E

    2017-01-01

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effec...

  17. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  18. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    Science.gov (United States)

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  19. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Regulation of protein homeostasis in neurodegenerative diseases : the role of coding and non-coding genes

    NARCIS (Netherlands)

    Alvarenga Fernandes Sin, Olga; Nollen, Ellen A. A.

    Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding,

  1. Regulation of Trafficking, Membrane Retention and Turnover of the Na+, HCO3- Co-Transporter NBCn1 in Epithelial Cells

    DEFF Research Database (Denmark)

    Olesen, Christina Wilkens

    normal conditions. Furthermore, we found that the small scaffolding protein RACK1 co-localizes with NBCn1 in membrane protrusions and is important for NBCn1 membrane stability. Collectively, the work in this thesis has contributed with new understanding of the mechanisms involved in cisplatin...

  2. Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes

    DEFF Research Database (Denmark)

    Nedergaard, Anders; Jespersen, Jakob G; Pingel, Jessica

    2012-01-01

    of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1...

  3. Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

    2009-11-15

    We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

  4. Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

    Science.gov (United States)

    Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

    2017-10-01

    Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  5. Intrinsically Disordered Segments Affect Protein Half-Life in the Cell and during Evolution

    NARCIS (Netherlands)

    Lee, R.T.J.G. van der; Lang, B.; Kruse, K.; Gsponer, J.; Groot, N.; Huynen, M.A.; Matouschek, A.; Fuxreiter, M.; Babu, M.M.

    2014-01-01

    Precise control of protein turnover is essential for cellular homeostasis. The ubiquitin-proteasome system is well established as a major regulator of protein degradation, but an understanding of how inherent structural features influence the lifetimes of proteins is lacking. We report that yeast,

  6. Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

    Directory of Open Access Journals (Sweden)

    Yosef Y Kuttner

    2013-04-01

    Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

  7. Engineered elastomeric proteins with dual elasticity can be controlled by a molecular regulator.

    Science.gov (United States)

    Cao, Yi; Li, Hongbin

    2008-08-01

    Elastomeric proteins are molecular springs that confer excellent mechanical properties to many biological tissues and biomaterials. Depending on the role performed by the tissue or biomaterial, elastomeric proteins can behave as molecular springs or shock absorbers. Here we combine single-molecule atomic force microscopy and protein engineering techniques to create elastomeric proteins that can switch between two distinct types of mechanical behaviour in response to the binding of a molecular regulator. The proteins are mechanically labile by design and behave as entropic springs with an elasticity that is governed by their configurational entropy. However, when a molecular regulator binds to the protein, it switches into a mechanically stable state and can act as a shock absorber. These engineered proteins effectively mimic and combine the two extreme forms of elastic behaviour found in natural elastomeric proteins, and thus represent a new type of smart nanomaterial that will find potential applications in nanomechanics and material sciences.

  8. Expression and Location of Glucose-regulated Protein 78 in Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    W Wang

    2014-04-01

    Full Text Available Objective: To know the role of glucose-regulated protein 78 (GRP78/BiP/HSPA5 in spermatogenesis and its expression and location in the testis and epididymis. Methods: Immunohistochemistry and Western blot were used to detect GRP78 location and expression in the testis and epididymis. Results: Glucose-regulated protein 78 was observed in spermatocytes, round spermatids and interstitial cells of the testis and in principal cells of the epididymis. Glucose-regulated protein 78 was first detected in the rat testis at postnatal day 14. Thereafter, the protein level increased gradually with age and was maintained at a high and stable state after postnatal day 28. In the rat, GRP78 was expressed in the principal cells but not in clear cells of the epididymis. Conclusion: Glucose-regulated protein 78 participates in the process of spermatogenesis.

  9. Fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75 interact with the CREC proteins, calumenin and reticulocalbin

    DEFF Research Database (Denmark)

    Hansen, Gry Aune Westergaard; Ludvigsen, Maja; Jacobsen, Christian

    2015-01-01

    Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted...

  10. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Science.gov (United States)

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M

    2013-01-01

    N-MYC down-regulated-like (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  11. N-MYC down-regulated-like proteins regulate meristem initiation by modulating auxin transport and MAX2 expression.

    Directory of Open Access Journals (Sweden)

    Yashwanti Mudgil

    Full Text Available N-MYC down-regulated-like (NDL proteins interact with the Gβ subunit (AGB1 of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.

  12. Protein turnover in the breast muscle of broiler chicks and studies addressing chlorine dioxide sanitation of hatching eggs, poultry leg problems and wheat middling diets for laying hens

    International Nuclear Information System (INIS)

    Patterson, P.H.

    1988-01-01

    Developmental changes occurred in breast muscle Ks measured by 14 C-tyrosine incorporation at 10, 16, 22 and 34 days of age. Protein synthesis rates decreased as the birds matures: 30 to 11.2%/d between 10 and 34 days of age. In a second study birds fed diets low in lysine or protein-energy had reduced fractional rates of protein synthesis and free tyrosine, branched chain and large neutral amino acid concentrations as compared to control birds the same body weight. Artificial weight loading and reduced dietary protein levels were used to study the effects of body weight on the severity of leg deformities in chicks and poults. Experiments investigating the practicality of wheat middlings as an alternate feedstuff for laying hens suggested that high levels in the diet will reduce egg production, feed conversion, hen livability and egg yolk color. Lastly, chlorine dioxide foam and dipping solutions were compared with formaldehyde fumigation for sanitizing hatching eggs

  13. A new technique for measuring protein turnover in the gut, liver and kidneys of lean and obese mice with [3H] glutamic acid

    International Nuclear Information System (INIS)

    Miller, B.G.; Grimble, R.F.; Taylor, T.G.

    1978-01-01

    Measurements have been made of the incorporation of an intraperitoneal injection of [ 3 H]glutamate into the protein of the gut, liver and kidney of lean and obese siblings of the genetically obese mouse. Recycling of the 3 H was minimized by using glutamate labelled at the C-2 position. Loss of label from the amino acid pool by transamination and deamination was rapid, with a half-life of 4 h. In tissue protein the amino acid showing the highest 3 H radioactivity was glutamate. The half-lives for protein synthesis and catabolism were calculated from the decay curves of both specific and total radioactivity of [ 3 H] glutamate in tissue protein. No significant differences were found between kidney, liver and gut in lean and obese mice. (author)

  14. Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

    Science.gov (United States)

    Tayou, Junior; Wang, Qiang; Jang, Geeng-Fu; Pronin, Alexey N; Orlandi, Cesare; Martemyanov, Kirill A; Crabb, John W; Slepak, Vladlen Z

    2016-04-22

    RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

    OpenAIRE

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

    2013-01-01

    Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the G? subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the presen...

  16. Structural Elements Regulating AAA+ Protein Quality Control Machines.

    Science.gov (United States)

    Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

    2017-01-01

    Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

  17. The effects of calcium regulation of endosperm reserve protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-15

    Jun 15, 2011 ... on barley endosperm protein mobilization during malting. Although, the site and ... fractionating head of the digesting vigreux column. The digest was ... growth and enormous reductions in malting loss (Ezeogu and Okolo ...

  18. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    OpenAIRE

    Treviño-Saldaña, Niria; García-Rivas, Gerardo

    2017-01-01

    Modulation of posttranslational modifications (PTMs), such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the developm...

  19. Importance of sulfide interaction with iron as regulator of the microbial community in biogas reactors and its effect on methanogenesis, volatile fatty acids turnover, and syntrophic long-chain fatty acids degradation.

    Science.gov (United States)

    Shakeri Yekta, Sepehr; Ziels, Ryan M; Björn, Annika; Skyllberg, Ulf; Ejlertsson, Jörgen; Karlsson, Anna; Svedlund, Matilda; Willén, Magnus; Svensson, Bo H

    2017-05-01

    The inhibitory effects of sulfide on microbial processes during anaerobic digestion have been widely addressed. However, other effects of sulfide are less explored, given that sulfide is a potential sulfur source for microorganisms and its high reactivity triggers a suit of abiotic reactions. We demonstrated that sulfide interaction with Fe regulates the dynamics and activities of microbial community during anaerobic digestion. This was manifested by the S:Fe molar ratio, whose increase adversely influenced the acetoclastic methanogens, Methanosaeta, and turnover of acetate. Dynamics of hydrogenotrophic methanogens, Methanoculleus and Methanobrevibacter, were presumably influenced by sulfide-induced changes in the partial pressure of hydrogen. Interestingly, conversion of the long-chain fatty acid (LCFA), oleate, to methane was enhanced together with the abundance of LCFA-degrading, β-oxidizing Syntrophomonas at an elevated S:Fe molar ratio. The results suggested that sulfur chemical speciation is a controlling factor for microbial community functions in anaerobic digestion processes. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Comparative proteomic analyses reveal that the regulators of G-protein signaling proteins regulate amino acid metabolism of the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Zhang, Haifeng; Ma, Hongyu; Xie, Xin; Ji, Jun; Dong, Yanhan; Du, Yan; Tang, Wei; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2014-11-01

    The rice blast fungus Magnaporthe oryzae encodes eight regulators of G-protein (GTP-binding protein) signaling (RGS) proteins MoRgs1-MoRgs8 that orchestrate the growth, asexual/sexual production, appressorium differentiation, and pathogenicity. To address the mechanisms by which MoRgs proteins function, we conducted a 2DE proteome study and identified 82 differentially expressed proteins by comparing five ∆Morgs mutants with wild-type Guy11 strain. We found that the abundances of eight amino acid (AA) biosynthesis or degradation associated proteins were markedly altered in five ∆Morgs mutants, indicating one of the main collective roles for the MoRgs proteins is to influence AA metabolism. We showed that MoRgs proteins have distinct roles in AA metabolism and nutrient responses from growth assays. In addition, we characterized MoLys20 (Lys is lysine), a homocitrate synthase, whose abundance was significantly decreased in the ∆Morgs mutants. The ∆Molys20 mutant is auxotrophic for lys and exogenous lys could partially rescue its auxotrophic defects. Deletion of MoLYS20 resulted in defects in conidiation and infection, as well as pathogenicity on rice. Overall, our results indicate that one of the critical roles for MoRgs proteins is to regulate AA metabolism, and that MoLys20 may be directly or indirectly regulated by MoRgs and participated in lys biosynthesis, thereby affecting fungal development and pathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

    Science.gov (United States)

    Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

    2005-07-01

    We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

  2. Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

    Directory of Open Access Journals (Sweden)

    Wenlin An

    Full Text Available Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI with a destabilizing domain (DD specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

  3. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

    Science.gov (United States)

    Church, George M.; Esvelt, Kevin; Mali, Prashant

    2017-03-07

    Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

  4. The Role of TSC Proteins in Regulating Cell Adhesion and Motility

    National Research Council Canada - National Science Library

    Krymskaya, Vera P

    2006-01-01

    The goal of this project was to define the molecular signaling mechanisms by which TSCI and TSC2 proteins regulate cell adhesion and motility as it relates to the genetic disorder tuberous sclerosis complex (TSC...

  5. DMPD: Regulation of innate immunity by suppressor of cytokine signaling (SOCS)proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18406369 Regulation of innate immunity by suppressor of cytokine signaling (SOCS)proteins...svg) (.html) (.csml) Show Regulation of innate immunity by suppressor of cytokine signaling (SOCS)proteins. ...PubmedID 18406369 Title Regulation of innate immunity by suppressor of cytokine signaling (SOCS)proteins

  6. BAG3: a multifaceted protein that regulates major cell pathways

    Science.gov (United States)

    Rosati, A; Graziano, V; De Laurenzi, V; Pascale, M; Turco, M C

    2011-01-01

    Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression. PMID:21472004

  7. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Examination of intracellular protein turnover under the influence of glucagon exemplified by the 14C-BNPP inhibitable liver carboxylesterases of diabetic rats

    International Nuclear Information System (INIS)

    Wagenmann, F.P.

    1982-01-01

    To-date, there are but few studies on the synthesis and degradation of cell proteins under the influence of exogenically supplied-hormones. The thesis aimed at undertaking such a study using membrane-linked non-specific liver carboxyl esterases under the influence of glucagon. The method applied allows to simultaneously measure both the content and the metabolism of carboxyl esterases. Test animals were 24 male Wistar rats which were turned into diabetics using streptozotocine. Following a single i.p. application of 10mg BNPP/kg body weight, the animals received, s.c. injections of 1-1.5 mg zinc protamine glucagon/kg bodyweight daily. Esterase content per gram of microsomal protein dropped to some 50% during the 5-day observation period, whereas total protein concentration of the microsome fraction was reduced but lightly. Such a drop in the esterase concentration in the liver could not be found in a 28-amimal control group which received neither streptozotocine nor zinc protamine glucagon. Average halflife of carboxyl esterases was 43 h in these animals whereas it was 73 h in the animals treated with glucagon. A mathematical model of protein degradation allowed to prove that the supraproportional decrease of esterase content and the marked prolongation of average halflife in glucagon-treated animals was mainly due to a restriction of the de-novo synthesis of esterasis. (orig./MG) [de

  9. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

  10. AKAP-scaffolding proteins and regulation of cardiac physiology

    Science.gov (United States)

    Mauban, JRH; O'Donnell, M; Warrier, S; Manni, S; Bond, M

    2009-01-01

    A kinase anchoring proteins (AKAPs) compose a growing list of diverse but functionally related proteins defined by their ability to bind to the regulatory subunit of protein kinase A. AKAPs perform an integral role in the spatiotemporal modulation of a multitude of cellular signaling pathways. This review highlights the extensive role of AKAPs in cardiac excitation/contraction coupling and cardiac physiology. The literature shows that particular AKAPs are involved in cardiac Ca2+ influx, release, re-uptake, and myocyte repolarization. Studies have also suggested roles for AKAPs in cardiac remodeling. Transgenic studies show functional effects of AKAPs, not only in the cardiovascular system, but in other organ systems as well. PMID:19364910

  11. Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

    OpenAIRE

    E. Ruchti; P.J. Roach; A.A. DePaoli-Roach; P.J. Magistretti; I. Allaman

    2016-01-01

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the ...

  12. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

  13. Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility*

    Science.gov (United States)

    Ayoub, Emily; Hall, Anita; Scott, Adam M.; Chagnon, Mélanie J.; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L.

    2013-01-01

    PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis. PMID:23897807

  14. Salary, Performance, and Superintendent Turnover

    Science.gov (United States)

    Grissom, Jason A.; Mitani, Hajime

    2016-01-01

    Purpose: Superintendent retention is an important goal for many school districts, yet the factors contributing to superintendent turnover are poorly understood. Most prior quantitative studies of superintendent turnover have relied on small, cross-sectional samples, limiting the evidence base. Utilizing longitudinal administrative records from…

  15. Modifications to the translational apparatus which affect the regulation of protein synthesis in sea urchin embryos

    International Nuclear Information System (INIS)

    Scalise, F.W.

    1988-01-01

    Protein synthesis can be regulated at a number of cellular levels. I have examined how modifications to specific components of the protein synthetic machinery are involved in regulating the efficiency of initiation of translation during early sea urchin embryogenesis. It is demonstrated that Ca 2+ concentrations exceeding 500 uM cause the inhibition of protein synthesis in cell-free translation lysates prepared from sea urchin embryos. Specific changes in the state of phosphorylation of at least 8 proteins occur during this Ca 2+ -mediated repression of translation. Analysis of these proteins has indicated that, unlike mammalian systems, there is no detectable level of Ca 2+ -dependent phosphorylation of the αsubunit eIF-2. Two of the proteins which do become phosphorylated in response to Ca 2+ are calmodulin and an isoelectric form of sea urchin eIF-4D. In addition, 2 proteins which share similarities with kinases involved in the regulation of protein synthesis in mammalian cells, also become phosphorylated. I have investigated the consequences of changes in eIF-4D during sea urchin embryogenesis because it has been proposed that a polyamine-mediated conversion of lysine to hypusine in this factor may enhance translational activity. It is demonstrated that [ 3 H] spermidine-derived radioactivity is incorporated into a number of proteins when sea urchin embryos are labeled in vivo, and that the pattern of individual proteins that become labeled changes over the course of the first 30 hr of development

  16. Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

    Science.gov (United States)

    Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

    2018-02-15

    The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Glucohexaose-induced protein phosphatase 2C regulates cell redox ...

    Indian Academy of Sciences (India)

    Q M Chen

    2018-02-13

    Feb 13, 2018 ... glucohexaose, CsPP2C80s play a positive regulatory role in process of ABA combined with ABA receptors ..... protein kinases (SnRKs) involve in the stress responses .... In this work, the endogenous ABA content increased.

  18. Developmental regulation of silk protein P25 in the silkworm ...

    Indian Academy of Sciences (India)

    tribpo

    anti-P25 sera were raised in rabbit and mice. The relative ... of B. mori. Couble et al (1983) identified a new mRNA species in the posterior ... quantitative changes in P25 protein level during development in B. mori. 2 . ..... physiology biochemistry and pharmacology (eds) G A Kerkut and L I Gilbert (New York: Pergamon.

  19. Regulation of triglyceride metabolism by angiopoietin-like proteins

    NARCIS (Netherlands)

    Mattijssen, F.B.J.; Kersten, A.H.

    2012-01-01

    asma triglyceride concentrations are determined by the balance between production of the triglyceride-rich lipoproteins VLDL and chylomicrons in liver and intestine, and their lipoprotein lipase-mediated clearance in peripheral tissues. In the last decade, the group of Angiopoietin-like proteins has

  20. Occupational stress and employee turnover.

    Science.gov (United States)

    Bridger, Robert S; Day, Andrea J; Morton, Kate

    2013-01-01

    Questionnaire data captured in January-March 2007 were examined in relation to turnover in males and females during the next five years. In general, most of the workplace stressors (such as role conflict or peer support) were not antecedents of turnover in any group. Junior personnel with psychological strain in 2007 had an increased risk of turnover in the next five years. Low commitment to the service in 2007 increased the odds of turnover in male and female juniors and in female officers. Female juniors with less effective skills for coping with stress and who exercised less frequently on a weekly basis were more likely to leave. An incidental finding was that the odds of turnover were three times greater in female officers with children than in female officers with no children. Stress management interventions focusing on effective coping and sports and exercise participation which are targeted appropriately may improve retention.

  1. The nuclear IκB family of proteins controls gene regulation and immune homeostasis.

    Science.gov (United States)

    MaruYama, Takashi

    2015-10-01

    The inhibitory IκB family of proteins is subdivided into two groups based on protein localization in the cytoplasm or in the nucleus. These proteins interact with NF-κB, a major transcription factor regulating the expression of many inflammatory cytokines, by modulating its transcriptional activity. However, nuclear IκB family proteins not only interact with NF-κB to change its transcriptional activity, but they also bind to chromatin and control gene expression. This review provides an overview of nuclear IκB family proteins and their role in immune homeostasis. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Managing voluntary turnover through challenging assignments

    NARCIS (Netherlands)

    Preenen, P.T.Y.; de Pater, I.E.; van Vianen, A.E.M.; Keijzer, L.

    2011-01-01

    This study examines employees’ challenging assignments as manageable means to reduce turnover intentions, job search behaviors, and voluntary turnover. Results indicate that challenging assignments are negatively related to turnover intentions and job search behaviors and that these relationships

  3. Managing voluntary turnover through challenging assignments

    NARCIS (Netherlands)

    Preenen, P.T.Y.; Pater, I.E. de; Vianen, A.E.M. van; Keijzer, L.

    2011-01-01

    This study examines employees' challenging assignments as manageable means to reduce turnover intentions, job search behaviors, and voluntary turnover. Results indicate that challenging assignments are negatively related to turnover intentions and job search behaviors and that these relationships

  4. Regulation of homologous recombination repair protein Rad51 by Ku70

    International Nuclear Information System (INIS)

    Du Liqing; Liu Qiang; Wang Yan; Xu Chang; Cao Jia; Fu Yue; Chen Fenghua; Fan Feiyue

    2013-01-01

    Objective: To explore the regulative effect of non-homologous end joining (NHEJ)protein Ku70 on homologous recombination repair protein Rad51, and to investigate the synergistic mechanism of homologous recombination repair in combination with NHEJ. Methods: Observed Rad51 protein expression after transfect Ku70 small interfering RNA or Ku70 plasmid DNA into tumor cells using Western blot. Results: Expression of Rad51 was obviously reduced after pretreated with Ku70 small interfering RNA. And with the increasing expression of Ku70 protein after transfection of Ku70 plasmid DNA PGCsi3.0-hKu70 into tumor cell lines, the Rad51 protein expression was increased. Conclusion: Ku70 protein has regulating effect on gene expression of Rad51, and it might participate in the collaboration between homologous recombination repair and NHEJ. (authors)

  5. Genetic regulation ofmethylation and IL1RL1-a protein levels in asthma

    NARCIS (Netherlands)

    Dijk, F Nicole; Xu, Chengjian; Melén, Erik; Carsin, Anne-Elie; Kumar, Asish; Nolte, Ilja M; Gruzieva, Olena; Pershagen, Goran; Grotenboer, Neomi S; Savenije, Olga E M; Antó, Josep Maria; Lavi, Iris; Dobaño, Carlota; Bousquet, Jean; van der Vlies, Pieter; van der Valk, Ralf J P; de Jongste, Johan C; Nawijn, Martijn C; Guerra, Stefano; Postma, Dirkje S; Koppelman, Gerard H

    2018-01-01

    Interleukin-1 receptor-like 1 (IL1RL1) is an important asthma gene. (Epi)genetic regulation ofIL1RL1protein expression has not been established. We assessed the association betweenIL1RL1single nucleotide polymorphisms (SNPs),IL1RL1methylation and serum IL1RL1-a protein levels, and aimed to identify

  6. The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

    NARCIS (Netherlands)

    Schmitz, Leanne J M; Klaassen, Remco V; Ruiperez-Alonso, Marta; Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J; Mansvelder, Huib D; Smit, August B; Spijker, Sabine; Verhage, Matthijs

    2017-01-01

    Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with

  7. RNA-binding proteins involved in post-transcriptional regulation in bacteria

    Directory of Open Access Journals (Sweden)

    Elke eVan Assche

    2015-03-01

    Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

  8. Regulation of activity of the yeast TATA-binding protein through intra ...

    Indian Academy of Sciences (India)

    Unknown

    Abbreviations used: BMH, Bismaleimidohexane; TBP, TATA-binding protein; yTBP, yeast TBP. J. Biosci. | Vol. ... Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar ..... simulations (Miaskeiwicz and Ornstein 1996). .... box binding protein (TBP): A molecular dynamics computa-.

  9. The role of polypyrimidine tract-binding proteins and other hnRNP proteins in plant splicing regulation

    Directory of Open Access Journals (Sweden)

    Andreas eWachter

    2012-05-01

    Full Text Available Alternative precursor mRNA splicing is a widespread phenomenon in multicellular eukaryotes and represents a major means for functional expansion of the transcriptome. While several recent studies have revealed an important link between splicing regulation and fundamental biological processes in plants, many important aspects, such as the underlying splicing regulatory mechanisms, are so far not well understood. Splicing decisions are in general based on a splicing code that is determined by the dynamic interplay of splicing-controlling factors and cis-regulatory elements. Several members of the group of heterogeneous nuclear ribonucleoprotein (hnRNP proteins are well-known regulators of splicing in animals and the comparatively few reports on some of their plant homologues revealed similar functions. This also applies to polypyrimidine tract-binding proteins (PTBs, a thoroughly investigated class of hnRNP proteins with splicing regulatory functions in both animals and plants. Further examples from plants are auto- and cross-regulatory splicing circuits of glycine-rich RNA-binding proteins (GRPs and splicing enhancement by oligouridylatebinding proteins. Besides their role in defining splice site choice, hnRNP proteins are also involved in multiple other steps of nucleic acid metabolism, highlighting the functional versatility of this group of proteins in higher eukaryotes.

  10. Microarray and Proteomic Analysis of Brassinosteroid- and Gibberellin-Regulated Gene and Protein Expression in Rice

    OpenAIRE

    Yang, Guangxiao; Komatsu, Setsuko

    2016-01-01

    Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze exp...

  11. Autoradiographic imaging of phosphoinositide turnover in the brain

    International Nuclear Information System (INIS)

    Hwang, P.M.; Bredt, D.S.; Snyder, S.H.

    1990-01-01

    With [ 3 H]cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product [ 3 H]cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways

  12. Body Characteristics, Dietary Protein and Body Weight Regulation

    DEFF Research Database (Denmark)

    Ankarfeldt, Mikkel Zøllner; Ängquist, Lars; Stocks, Tanja

    2014-01-01

    between greater protein intake and weight gain. In both types of studies the results are based on average weight changes, and show considerable diversity in both directions. This study investigates whether the discrepancy in the evidence could be due to recruitment of overweight and obese individuals...... with greater body mass index and waist circumference were analyzed. Selecting subsets of large-scale observational cohort studies with similar characteristics as participants in clinical trials may reconcile the otherwise conflicting results....

  13. Regulation, cell differentiation and protein-based inheritance.

    Science.gov (United States)

    Malagnac, Fabienne; Silar, Philippe

    2006-11-01

    Recent research using fungi as models provide new insight into the ability of regulatory networks to generate cellular states that are sufficiently stable to be faithfully transmitted to daughter cells, thereby generating epigenetic inheritance. Such protein-based inheritance is driven by infectious factors endowed with properties usually displayed by prions. We emphasize the contribution of regulatory networks to the emerging properties displayed by cells.

  14. Mitochondrial fission proteins regulate programmed cell death in yeast

    OpenAIRE

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J.; Qi, Bing; Pevsner, Jonathan; McCaffery, J. Michael; Hill, R. Blake; Basañez, Gorka; Hardwick, J. Marie

    2004-01-01

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we fo...

  15. Structural aspects of protein kinase ASK1 regulation

    Czech Academy of Sciences Publication Activity Database

    Obšil, Tomáš; Obšilová, Veronika

    2017-01-01

    Roč. 66, 1 Dec (2017), s. 31-36 ISSN 2212-4926 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ASK1 kinase * apoptosis * thioredoxin * 14-3-3 protein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  16. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  17. Src protein-tyrosine kinase structure and regulation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2004-01-01

    Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

  18. Regulation of amyloid precursor protein processing by its KFERQ motif.

    Science.gov (United States)

    Park, Ji-Seon; Kim, Dong-Hou; Yoon, Seung-Yong

    2016-06-01

    Understanding of trafficking, processing, and degradation mechanisms of amyloid precursor protein (APP) is important because APP can be processed to produce β-amyloid (Aβ), a key pathogenic molecule in Alzheimer's disease (AD). Here, we found that APP contains KFERQ motif at its C-terminus, a consensus sequence for chaperone-mediated autophagy (CMA) or microautophagy which are another types of autophagy for degradation of pathogenic molecules in neurodegenerative diseases. Deletion of KFERQ in APP increased C-terminal fragments (CTFs) and secreted N-terminal fragments of APP and kept it away from lysosomes. KFERQ deletion did not abolish the interaction of APP or its cleaved products with heat shock cognate protein 70 (Hsc70), a protein necessary for CMA or microautophagy. These findings suggest that KFERQ motif is important for normal processing and degradation of APP to preclude the accumulation of APP-CTFs although it may not be important for CMA or microautophagy. [BMB Reports 2016; 49(6): 337-342].

  19. EBF proteins participate in transcriptional regulation of Xenopus muscle development.

    Science.gov (United States)

    Green, Yangsook Song; Vetter, Monica L

    2011-10-01

    EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

    Science.gov (United States)

    Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

    2013-01-14

    The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-11-15

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

  2. Regulator of G-Protein Signaling 7 Regulates Reward Behavior by Controlling Opioid Signaling in the Striatum.

    Science.gov (United States)

    Sutton, Laurie P; Ostrovskaya, Olga; Dao, Maria; Xie, Keqiang; Orlandi, Cesare; Smith, Roy; Wee, Sunmee; Martemyanov, Kirill A

    2016-08-01

    Morphine mediates its euphoric and analgesic effects by acting on the μ-opioid receptor (MOR). MOR belongs to the family of G-protein coupled receptors whose signaling efficiency is controlled by the regulator of G-protein signaling (RGS) proteins. Our understanding of the molecular diversity of RGS proteins that control MOR signaling, their circuit specific actions, and underlying cellular mechanisms is very limited. We used genetic approaches to ablate regulator of G-protein signaling 7 (RGS7) both globally and in specific neuronal populations. We used conditioned place preference and self-administration paradigms to examine reward-related behavior and a battery of tests to assess analgesia, tolerance, and physical dependence to morphine. Electrophysiology approaches were applied to investigate the impact of RGS7 on morphine-induced alterations in neuronal excitability and plasticity of glutamatergic synapses. At least three animals were used for each assessment. Elimination of RGS7 enhanced reward, increased analgesia, delayed tolerance, and heightened withdrawal in response to morphine administration. RGS7 in striatal neurons was selectively responsible for determining the sensitivity of rewarding and reinforcing behaviors to morphine without affecting analgesia, tolerance, and withdrawal. In contrast, deletion of RGS7 in dopaminergic neurons did not influence morphine reward. RGS7 exerted its effects by controlling morphine-induced changes in excitability of medium spiny neurons in nucleus accumbens and gating the compositional plasticity of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors. This study identifies RGS7 as a novel regulator of MOR signaling by dissecting its circuit specific actions and pinpointing its role in regulating morphine reward by controlling the activity of nucleus accumbens neurons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  3. CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

    Science.gov (United States)

    Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

    1999-09-10

    Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

  4. Hormone response element binding proteins: novel regulators of vitamin D and estrogen signaling.

    Science.gov (United States)

    Lisse, Thomas S; Hewison, Martin; Adams, John S

    2011-03-01

    Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as "vitamin D or estrogen response element-binding proteins", behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vitamin D and estrogen resistances. This set of interactors belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of previously known pre-mRNA-interacting proteins. This review provides new insights into the mechanism by which these novel regulators of signaling and metabolism can act to regulate responses to vitamin D and estrogen. In addition the review also describes other molecules that are known to influence nuclear receptor signaling through interaction with hormone response elements. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Jacob B; te Riele, Hein; Kristiansen, Karsten

    2004-01-01

    the major energy store and brown adipocytes being potent energy-dissipaters through thermogenesis. Yet, little is known about factors differentially regulating the formation of white and brown fat cells. Members of the retinoblastoma protein family (pRB, p107, p130) have been implicated in the regulation...... of adipocyte differentiation, and expression and phosphorylation of the three retinoblastoma family proteins oscillate in a characteristic manner during differentiation of the white preadipocyte cell line 3T3-L1. We have recently demonstrated a surprising function of the retinoblastoma protein...... in the regulation of white versus brown adipocyte differentiation in vitro and possibly in vivo. Here we summarize the current knowledge on the retinoblastoma protein in fat cells, with particular emphasis on its potential role in adipocyte lineage commitment and differentiation....

  6. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans.

    Science.gov (United States)

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-21

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  8. The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase.

    Directory of Open Access Journals (Sweden)

    M Qaiser Fatmi

    Full Text Available The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS. TRPS uses a set of α/β-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the α/β-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

  9. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    ;1 in Arabidopsis. That led to the discovery that tip4;1 is gametophytic lethal- gene essential for normal seed set. ICP-MS analyses of the elemental composition of tip4;1 heterozygous T-DNA insert mutant plants and 35S::TIP4;1 over-expression plants indicate that AtTIP4;1 has a role in arsenic distribution...... inorganic forms of arsenic in the environment, can be taken up by plants and thus enter the food chain. Once inside the root cells, As(V) is reduced to As(III) which is then extruded to the soil solution or bound to phytochelatins (PCs) and transported to the vacuole in an effort to accomplish...... detoxification. Plant Noduline 26-like Intrinsic Proteins (NIPs) can channel As(III) and consequently influence the detoxification process. The role of the Tonoplast Intrinsic Proteins (TIPs) in As(III) detoxification remains to be clarified, yet TIPs could have an impact on As(III) accumulation in plant cell...

  10. How Employee Turnover Affects Productivity

    DEFF Research Database (Denmark)

    Eriksen, Bo

    Research on employee turnover suggests that turnover results in negative organization-level outcomes. This paper provides a firm-level analysis of the impact of the in- and outflows of human resources on productivity and how the presence of organizational slack resources moderates the effects...... moderate this effect so that the negative consequences of employee turnover are less severe for larger, older and capital intensive firms. These moderating variables indicate the presence of slack resources in the firm, and thus that the accumulation of slack reduces the efficiency losses from employee...

  11. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... on protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  12. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  13. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells

    International Nuclear Information System (INIS)

    Gao Zhihua; Jin Jinghua; Yang Jun; Yu Yingnian

    2004-01-01

    Proteomic analysis, which combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), is an important approach to screen proteins responsive to specific stimuli. Benzo[a]pyrene (B[a]P), a prototype of polycyclic hydrocarbons (PAHs), is a potent procarcinogen generated from the combustion of fossil fuel and cigarette smoke. To further probe the molecular mechanism of mutagenesis and carcinogenesis, and to find potential molecular markers involved in cellular responses to B[a]P exposure, we performed proteomic analysis of whole cellular proteins in human amnion epithelial cells after B[a]P-treatment. Image visualization and statistical analysis indicated that more than 40 proteins showed significant changes following B[a]P-treatment (P<0.05). Among them, 20 proteins existed only in the control groups, while six were only present in B[a]P-treated cells. In addition, the expression of 10 proteins increased whereas 11 decreased after B[a]P-treatment. These proteins were subjected to in-gel tryptic digestion followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. Using peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified 22 proteins. Most of these proteins have unknown functions and have not been previously connected to a response to B[a]P exposure. To further annotate the characteristics of these proteins, GOblet analysis was carried out and results indicated that they were involved in multiple biological processes including regulation of transcription, cell proliferation, cell aging and other processes. However, expression changes were noted in a number of transcription regulators, including eight zinc finger proteins as well as SNF2L1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1), which is closely linked to the chromatin remodeling process. These data may provide new clues to further understand the implication of

  14. Acid-regulated proteins induced by Streptococcus mutans and other oral bacteria during acid shock.

    Science.gov (United States)

    Hamilton, I R; Svensäter, G

    1998-10-01

    Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the

  15. Tendon 'turnover lengthening' technique.

    Science.gov (United States)

    Cerovac, S; Miranda, B H

    2013-11-01

    Tendon defect reconstruction is amongst the most technically challenging areas in hand surgery. Tendon substance deficiency reconstruction techniques include lengthening, grafting, two-stage reconstruction and tendon transfers, however each is associated with unique challenges over and above direct repair. We describe a novel 'turnover lengthening' technique for hand tendons that has successfully been applied to the repair of several cases, including a case of attritional flexor and traumatic extensor tendon rupture in two presented patients where primary tenorrhaphy was not possible. In both cases a good post-operative outcome was achieved, as the patients were happy having returned back to normal activities of daily living such that they were discharged 12 weeks post-operatively. Our technique avoids the additional morbidity and complications associated with grafting, transfers and two stage reconstructions. It is quick, simple and reproducible for defects not exceeding 3-4 cm, provides a means of immediate one stage reconstruction, no secondary donor site morbidity and does not compromise salvage by tendon transfer and/or two-stage reconstruction in cases of failure. To our knowledge no such technique has been previously been described to reconstruct such hand tendon defects. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  16. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding.

    Science.gov (United States)

    Kleiman, Laura B; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A; Sorger, Peter K

    2011-09-02

    Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  18. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

    2009-01-01

    of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

  19. High bone turnover in Irish professional jockeys.

    LENUS (Irish Health Repository)

    Waldron-Lynch, F

    2012-02-01

    SUMMARY: Professional jockeys are routinely exposed to high impact trauma and sustain fractures frequently. We found that jockeys restrict their caloric intake in order to maintain regulation weights, and that bone turnover is high. There are significant health and safety implications for the racing industry. INTRODUCTION: Professional jockeys routinely sustain fractures from high impact falls. Jockeys maintain a low percentage body fat and a low body mass index (BMI) to achieve low weight targets in order to race. We evaluated dietary habits and bone metabolism in jockeys. METHODS: Bone mineral density (BMD) was measured in 27 male jockeys of the 144 jockeys licensed in Ireland. Fourteen (52%) had BMD T score below -1.0, of whom 12 consented to clinical review, nutritional survey, endocrine studies, and bone turnover markers (BTM). BTM were compared to age- and sex-matched controls (n = 16). RESULTS: BMI was 20.6 +\\/- 1.7 kg\\/m(2); previous fracture frequency was 3.2 +\\/- 2.0 per rider. All had normal endocrine axes. The jockeys\\' diet as determined by a 7-day dietary recall was deficient in energy, calcium, and vitamin D intake. Compared with the control group, the jockey group had evidence of increased bone turnover. CONCLUSIONS: A substantial proportion of the professional jockeys in Ireland have low-normal BMD, low BMI, and high bone turnover that may result from weight and dietary restrictions. These factors seem to have a deleterious effect on their bone health and predispose the jockeys to a high fracture risk that should be remediated.

  20. Isolation of nuclear proteins from flax (Linum usitatissimum L. seed coats for gene expression regulation studies

    Directory of Open Access Journals (Sweden)

    Renouard Sullivan

    2012-01-01

    Full Text Available Abstract Background While seed biology is well characterized and numerous studies have focused on this subject over the past years, the regulation of seed coat development and metabolism is for the most part still non-elucidated. It is well known that the seed coat has an essential role in seed development and its features are associated with important agronomical traits. It also constitutes a rich source of valuable compounds such as pharmaceuticals. Most of the cell genetic material is contained in the nucleus; therefore nuclear proteins constitute a major actor for gene expression regulation. Isolation of nuclear proteins responsible for specific seed coat expression is an important prerequisite for understanding seed coat metabolism and development. The extraction of nuclear proteins may be problematic due to the presence of specific components that can interfere with the extraction process. The seed coat is a rich source of mucilage and phenolics, which are good examples of these hindering compounds. Findings In the present study, we propose an optimized nuclear protein extraction protocol able to provide nuclear proteins from flax seed coat without contaminants and sufficient yield and quality for their use in transcriptional gene expression regulation by gel shift experiments. Conclusions Routinely, around 250 μg of nuclear proteins per gram of fresh weight were extracted from immature flax seed coats. The isolation protocol described hereafter may serve as an effective tool for gene expression regulation and seed coat-focused proteomics studies.

  1. Collagen turnover after tibial fractures

    DEFF Research Database (Denmark)

    Joerring, S; Krogsgaard, M; Wilbek, H

    1994-01-01

    Collagen turnover after tibial fractures was examined in 16 patients with fracture of the tibial diaphysis and in 8 patients with fracture in the tibial condyle area by measuring sequential changes in serological markers of turnover of types I and III collagen for up to 26 weeks after fracture....... The markers were the carboxy-terminal extension peptide of type I procollagen (PICP), the amino-terminal extension peptide of type III procollagen (PIIINP), and the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP). The latter is a new serum marker of degradation of type I...... collagen. A group comparison showed characteristic sequential changes in the turnover of types I and III collagen in fractures of the tibial diaphysis and tibial condyles. The turnover of type III collagen reached a maximum after 2 weeks in both groups. The synthesis of type I collagen reached a maximum...

  2. SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

    Science.gov (United States)

    Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

    2011-02-04

    SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

  3. The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

    Science.gov (United States)

    Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

    2016-07-01

    In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. © 2015 Institute of Botany, Chinese Academy of Sciences.

  4. Regulating the ethylene response of a plant by modulation of F-box proteins

    Science.gov (United States)

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2014-01-07

    The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

  5. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    Science.gov (United States)

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. Copyright © 2016 the American Physiological Society.

  6. Amyloid beta precursor protein regulates male sexual behavior.

    Science.gov (United States)

    Park, Jin Ho; Bonthius, Paul J; Tsai, Houng-Wei; Bekiranov, Stefan; Rissman, Emilie F

    2010-07-28

    Sexual behavior is variable between individuals, ranging from celibacy to sexual addictions. Within normal populations of individual men, ranging from young to middle aged, testosterone levels do not correlate with libido. To study the genetic mechanisms that contribute to individual differences in male sexual behavior, we used hybrid B6D2F1 male mice, which are a cross between two common inbred strains (C57BL/6J and DBA/2J). Unlike most laboratory rodent species in which male sexual behavior is highly dependent upon gonadal steroids, sexual behavior in a large proportion of these hybrid male mice after castration is independent of gonadal steroid hormones and their receptors; thus, we have the ability to discover novel genes involved in this behavior. Gene expression arrays, validation of gene candidates, and transgenic mice that overexpress one of the genes of interest were used to reveal genes involved in maintenance of male sexual behavior. Several genes related to neuroprotection and neurodegeneration were differentially expressed in the hypothalamus of males that continued to mate after castration. Male mice overexpressing the human form of one of these candidate genes, amyloid beta precursor protein (APP), displayed enhanced sexual behavior before castration and maintained sexual activity for a longer duration after castration compared with controls. Our results reveal a novel and unexpected relationship between APP and male sexual behavior. We speculate that declining APP during normal aging in males may contribute to the loss of sexual function.

  7. Modulation of PML protein expression regulates JCV infection

    International Nuclear Information System (INIS)

    Gasparovic, Megan L.; Maginnis, Melissa S.; O'Hara, Bethany A.; Dugan, Aisling S.; Atwood, Walter J.

    2009-01-01

    JC virus (JCV) is a human polyomavirus that infects the majority of the human population worldwide. It is responsible for the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy. JCV binds to cells using the serotonin receptor 5-HT 2A R and α(2-6)- or α(2-3)-linked sialic acid. It enters cells using clathrin-dependent endocytosis and traffics to the early endosome and possibly to the endoplasmic reticulum. Viral DNA is then delivered to the nucleus where transcription, replication, and assembly of progeny occur. We found that the early regulatory protein large T antigen accumulates in microdomains in the nucleus adjacent to ND-10 or PML domains. This observation prompted us to explore the role of these domains in JCV infection. We found that a reduction of nuclear PML enhanced virus infection and that an increase in nuclear PML reduced infection. Infection with JCV did not directly modulate nuclear levels of PML but our data indicate that a host response involving interferon beta is likely to restrict virus infection by increasing nuclear PML.

  8. Granule protein processing and regulated secretion in neutrophils

    Directory of Open Access Journals (Sweden)

    Avinash eSheshechalam

    2014-09-01

    Full Text Available Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

  9. [Turnover Experience of Male Nurses].

    Science.gov (United States)

    Kim, Hyunsu; Lee, Jeongseop

    2017-02-01

    The aim of this study was to identify turnover experiences of men in nursing and to derive a substantive theory on the turnover experience of men who are nurses. Data were collected through in-depth interviews with 13 men who had worked as a nurse for 1 year or more, and had a turnover experience during that period. Collected data were analyzed on the basis of Strauss and Corbin's grounded theory. The core category in the turnover experiences of the respondents was 'seeking a stable place for me'. In the analysis of the core category, types of 'contentment', 'seeking', 'survival' and 'confusion' were identified. The sequential stages of these nurses' turnover experience were 'confrontation', 'incertitude', 'retrying' and 'realization'. However, when a problem arose in the process, they returned to the stage of confusion. Thus, these stages could occur in a circular fashion. These findings provide a deep understanding of the turnover experience of men in nursing and offers new information about how they adapt to nursing practice. The findings should be useful as foundational data for men who hope to become nurses and also for managers responsible for nurses who are men. © 2017 Korean Society of Nursing Science

  10. Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

    Science.gov (United States)

    Pawson, Tony; Kofler, Michael

    2009-04-01

    The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

  11. Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

    Science.gov (United States)

    Singh, Ajay; Haldar, Swati; Horback, Katharine; Tom, Cynthia; Zhou, Lan; Meyerson, Howard; Singh, Neena

    2017-01-01

    Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP−/−). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP−/− mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP−/− mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP−/− mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP−/− BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. PMID:23478311

  12. Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

    Science.gov (United States)

    Scherer, Stephanie L; Cain, Matthew D; Kanai, Stanley M; Kaltenbronn, Kevin M; Blumer, Kendall J

    2017-06-16

    The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function. © 2017 by

  13. Interaction of a plant pseudo-response regulator with a calmodulin-like protein

    Energy Technology Data Exchange (ETDEWEB)

    Perochon, Alexandre; Dieterle, Stefan; Pouzet, Cecile; Aldon, Didier; Galaud, Jean-Philippe [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France); Ranty, Benoit, E-mail: ranty@scsv.ups-tlse.fr [UMR 5546 CNRS/Universite Toulouse 3, Pole de Biotechnologie vegetale, BP 42617 Auzeville, 31326 Castanet-Tolosan cedex (France)

    2010-08-06

    Research highlights: {yields} The pseudo-response regulator PRR2 specifically binds CML9, a calmodulin-like protein {yields} The interaction is confirmed in plant cell nuclei {yields} The interaction requires an intact PRR2 protein. -- Abstract: Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

  14. Interaction of a plant pseudo-response regulator with a calmodulin-like protein

    International Nuclear Information System (INIS)

    Perochon, Alexandre; Dieterle, Stefan; Pouzet, Cecile; Aldon, Didier; Galaud, Jean-Philippe; Ranty, Benoit

    2010-01-01

    Research highlights: → The pseudo-response regulator PRR2 specifically binds CML9, a calmodulin-like protein → The interaction is confirmed in plant cell nuclei → The interaction requires an intact PRR2 protein. -- Abstract: Calmodulin (CaM) plays a crucial role in the regulation of diverse cellular processes by modulating the activities of numerous target proteins. Plants possess an extended CaM family including numerous CaM-like proteins (CMLs), most of which appear to be unique to plants. We previously demonstrated a role for CML9 in abiotic stress tolerance and seed germination in Arabidopsis thaliana. We report here the isolation of PRR2, a pseudo-response regulator as a CML9 interacting protein by screening an expression library prepared from Arabidopsis seedlings with CML9 as bait in a yeast two-hybrid system. PRR2 is similar to the response regulators of the two-component system, but lacks the invariant residue required for phosphorylation by which response regulators switch their output response, suggesting the existence of alternative regulatory mechanisms. PRR2 was found to bind CML9 and closely related CMLs but not a canonical CaM. Mapping analyses indicate that an almost complete form of PRR2 is required for interaction with CML9, suggesting a recognition mode different from the classical CaM-target peptide complex. PRR2 contains several features that are typical of transcription factors, including a GARP DNA recognition domain, a Pro-rich region and a Golden C-terminal box. PRR2 and CML9 as fusion proteins with fluorescent tags co-localized in the nucleus of plant cells, and their interaction in the nuclear compartment was validated in planta by using a fluorophore-tagged protein interaction assay. These findings suggest that binding of PRR2 to CML9 may be an important mechanism to modulate the physiological role of this transcription factor in plants.

  15. Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

    DEFF Research Database (Denmark)

    Arevalo-Ferro, C.; Hentzer, Morten; Reil, G.

    2003-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely......-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and Has......Ap, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type...

  16. Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

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    Pooja Singhmar

    Full Text Available Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

  17. Regulator of G-protein signaling - 5 (RGS5 is a novel repressor of hedgehog signaling.

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    William M Mahoney

    Full Text Available Hedgehog (Hh signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc and smoothened (Smo. Recent studies identify Smo as a G-protein coupled receptor (GPCR-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP, we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

  18. Topology and Oligomerization of Mono- and Oligomeric Proteins Regulate Their Half-Lives in the Cell.

    Science.gov (United States)

    Mallik, Saurav; Kundu, Sudip

    2018-06-05

    To find additional structural constraints (besides disordered segments) that regulate protein half-life in the cell, we herein assess the influence of native topology of monomeric and sequestration of oligomeric proteins into multimeric complexes in yeast, human, and mouse. Native topology acts as a molecular marker of globular protein's mechanical resistance and consequently captures their half-life variations on genome scale. Sequestration into multimeric complexes elongates oligomeric protein half-life in the cell, presumably by burying ubiquitinoylation sites and disordered segments required for proteasomal recognition. The latter effect is stronger for proteins associated with multiple complexes and for those binding early during complex self-assembly, including proteins that oligomerize with large proportions of surface buried. After gene duplication, diversification of topology and sequestration into non-identical sets of complexes alter half-lives of paralogous proteins during the course of evolution. Thus, native topology and sequestration into multimeric complexes reflect designing principles of proteins to regulate their half-lives. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    Science.gov (United States)

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

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  20. Structural analysis of DNA–protein complexes regulating the restriction–modification system Esp1396I

    International Nuclear Information System (INIS)

    Martin, Richard N. A.; McGeehan, John E.; Ball, Neil J.; Streeter, Simon D.; Thresh, Sarah-Jane; Kneale, G. G.

    2013-01-01

    Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation. The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex

  1. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Collingwood, S.A.; Ingolfsson, H.I.

    2010-01-01

    with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated...... with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids...... channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli....

  2. A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

    Science.gov (United States)

    Perego, M

    1997-08-05

    The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

  3. Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators

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    Shucai eWang

    2016-03-01

    Full Text Available OVATE FAMILY PROTEINS (OFPs are a class of proteins with a conserved OVATE domain. OVATE protein was first identified in tomato as a key regulator of fruit shape. OFPs are plant-specific proteins that are widely distributed in the plant kingdom including mosses and lycophytes. Transcriptional activity analysis of Arabidopsis OFPs (AtOFPs in protoplasts suggests that they act as transcription repressors. Functional characterization of OFPs from different plant species including Arabidopsis, rice, tomato, pepper and banana suggests that OFPs regulate multiple aspects of plant growth and development, which is likely achieved by interacting with different types of transcription factors including the KNOX and BELL classes, and/or directly regulating the expression of target genes such as Gibberellin 20 oxidase (GA20ox. Here, we examine how OVATE was originally identified, summarize recent progress in elucidation of the roles of OFPs in regulating plant growth and development, and describe possible mechanisms underpinning this regulation. Finally, we review potential new research directions that could shed additional light on the functional biology of OFPs in plants.

  4. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

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    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  5. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

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    Yeon Jeong Kim

    Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

  6. Business Ethics & Employee Turnover: CAFE Matrix

    OpenAIRE

    Sapovadia, Vrajlal; Patel, Sweta

    2013-01-01

    Abstract: Business ethics is in discussion for its importance universally, so is the employee turnover in business. Unethical practices are unwanted, so is the high employee turnover. Unethical practices and high employee turnover in business is ubiquitous. No consensus exists on defining ethics. Employee turnover is well defined, but there is no consensus on when employee turnover is disadvantageous for the company. The Golden Rule or ethic of reciprocity, a maxim states that either ...

  7. Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

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    Gry Aune Westergaard Hansen

    Full Text Available Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 μM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

  8. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

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    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  9. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

    Science.gov (United States)

    Zhang, Hua; Song, Lei; Cong, Haolong; Tien, Po

    2015-10-01

    Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially

  10. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

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    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  11. Dicer-like Proteins Regulate the Growth, Conidiation, and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

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    Qiannan Wang

    2018-01-01

    Full Text Available Colletotrichum gloeosporioides from Hevea brasiliensis is the hemibiotrophic fungi which could cause anthracnose in rubber trees. Dicer like proteins (DCL were the core enzymes for generation of small RNAs. In the present study, the knocking-out mutants of two dicer like proteins encoding genes of C. gloeosporioides were constructed; and functions of two proteins were investigated. The results showed that DCL play important roles in regulating the growth, conidiation and pathogenicity of C. gloeosporioides; and there is a functional redundancy between DCL1 and DCL2. Microscopy analysis and DAB staining revealed that loss of penetration ability into the host cells, instead of the decreased growth rate, was the main cause for the impaired pathogenicity of the ΔDcl1ΔDcl2 double mutant. Proteomics analysis suggested that DCL proteins affected the expression of functional proteins to regulating multiple biological processes of C. gloeosporioides. These data lead to a better understanding of the functions of DCL proteins in regulating the development and pathogenesis of C. gloeosporioides.

  12. The nuclear import of ribosomal proteins is regulated by mTOR

    Science.gov (United States)

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  13. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

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    Kai Yang

    2014-02-01

    Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

  14. The SCF ubiquitin ligase Slimb controls Nerfin-1 turnover in Drosophila.

    Science.gov (United States)

    Lin, Xiaohui; Wang, Feng; Li, Yuanpei; Zhai, Chaojun; Wang, Guiping; Zhang, Xiaoting; Gao, Yang; Yi, Tao; Sun, Dan; Wu, Shian

    2018-01-01

    The C2H2 type zinc-finger transcription factor Nerfin-1 expresses dominantly in Drosophila nervous system and plays an important role in early axon guidance decisions and preventing neurons dedifferentiation. Recently, increasing reports indicated that INSM1 (homologue to nerfin-1 in mammals) is a useful marker for prognosis of neuroendocrine tumors. The dynamic expression of Nerfin-1 is regulated post-transcriptionally by multiple microRNAs; however, its post-translational regulation is still unclear. Here we showed that the protein turnover of Nerfin-1 is regulated by Slimb, the substrate adaptor of SCF Slimb ubiquitin ligase complex. Mechanistically, Slimb associates with Nerfin-1 and promotes it ubiquitination and degradation in Drosophila S2R + cells. Furthermore, we determined that the C-terminal half of Nerfin-1 (Nerfin-1 CT ) is required for its binding to Slimb. Genetic epistasis assays showed that Slimb misexpression antagonizes, while knock-down enhances the activity of Nerfin-1 CT in Drosophila eyes. Our data revealed a new link to understand the underlying mechanism for Nerfin-1 turnover in post-translational level, and provided useful insights in animal development and disease treatment by manipulating the activity of Slimb and Nerfin-1. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

    Science.gov (United States)

    Rigamonti, Elena; Parolini, Cinzia; Marchesi, Marta; Diani, Erika; Brambilla, Stefano; Sirtori, Cesare R; Chiesa, Giulia

    2010-05-01

    Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (pPea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (ppea protein-fed rats than in rats fed casein (ppea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.

  16. Iron-regulated proteins (IRPS of leptospira biflexa serovar Patoc strain Patoc I

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    Sritharan M

    2004-01-01

    Full Text Available BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N′-diacetic acid (EDDA and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA and the second method involved the addition of iron at 0.02 µg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 µg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase, periplasmic space (aqueous phase and the protoplasmic cylinder (cell pellet. The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 µg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 µg Fe/mL. The blue CAS agar plates with top agar containing 0.02µg Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation

  17. The homeodomain protein ladybird late regulates synthesis of milk proteins during pregnancy in the tsetse fly (Glossina morsitans.

    Directory of Open Access Journals (Sweden)

    Geoffrey M Attardo

    2014-04-01

    Full Text Available Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina, the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. Here we identify and delineate the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1 by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This

  18. Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

    Science.gov (United States)

    Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

    2017-07-01

    Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

    Science.gov (United States)

    Chichger, Havovi; Braza, Julie; Duong, Huetran; Harrington, Elizabeth O

    2015-06-01

    Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients

  20. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    Science.gov (United States)

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

  1. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    Directory of Open Access Journals (Sweden)

    Jenkins Dafyd J

    2008-01-01

    Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

  2. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    OpenAIRE

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in tr...

  3. Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios.

    Science.gov (United States)

    Lu, Renfei; Osei-Adjei, George; Huang, Xinxiang; Zhang, Yiquan

    2018-03-01

    Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

  4. Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin

    DEFF Research Database (Denmark)

    Wu, Xunwei; Quondamatteo, Fabio; Lefever, Tine

    2006-01-01

    for differentiation of skin progenitor cells into HF lineage and that it regulates the turnover of beta-catenin. In the absence of Cdc42, degradation of beta-catenin was increased corresponding to a decreased phosphorylation of GSK3beta at Ser 9 and an increased phosphorylation of axin, which is known to be required...... for binding of beta-catenin to the degradation machinery. Cdc42-mediated regulation of beta-catenin turnover was completely dependent on PKCzeta, which associated with Cdc42, Par6, and Par3. These data suggest that Cdc42 regulation of beta-catenin turnover is important for terminal differentiation of HF...

  5. Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

    Science.gov (United States)

    Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

    2013-11-01

    Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

  6. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza; Jung, Sung Yun; Liu, Dou; Su, Bing; Qin, Jun

    2012-01-01

    capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit

  7. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  8. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

    Directory of Open Access Journals (Sweden)

    Naofumi Miwa

    2015-05-01

    Full Text Available Protein-carbohydrate interaction regulates multiple important processes during fertilization, an essential biological event where individual gametes undergo intercellular recognition to fuse and generate a zygote. In the mammalian female reproductive tract, sperm temporarily adhere to the oviductal epithelium via the complementary interaction between carbohydrate-binding proteins on the sperm membrane and carbohydrates on the oviductal cells. After detachment from the oviductal epithelium at the appropriate time point following ovulation, sperm migrate and occasionally bind to the extracellular matrix, called the zona pellucida (ZP, which surrounds the egg, thereafter undergoing the exocytotic acrosomal reaction to penetrate the envelope and to reach the egg plasma membrane. This sperm-ZP interaction also involves the direct interaction between sperm carbohydrate-binding proteins and carbohydrates within the ZP, most of which have been conserved across divergent species from mammals to amphibians and echinoderms. This review focuses on the carbohydrate-mediated interaction of sperm with the female reproductive tract, mainly the interaction between sperm and the ZP, and introduces the fertilization-suppressive action of dicalcin, a Xenopus laevis ZP protein-associated protein. The action of dicalcin correlates significantly with a dicalcin-dependent change in the lectin-staining pattern within the ZP, suggesting a unique role of dicalcin as an inherent protein that is capable of regulating the affinity between the lectin and oligosaccharides attached on its target glycoprotein.

  9. Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer.

    Science.gov (United States)

    Roberts, David D; Kaur, Sukhbir; Isenberg, Jeffrey S

    2017-10-20

    In contrast to structural elements of the extracellular matrix, matricellular proteins appear transiently during development and injury responses, but their sustained expression can contribute to chronic disease. Through interactions with other matrix components and specific cell surface receptors, matricellular proteins regulate multiple signaling pathways, including those mediated by reactive oxygen and nitrogen species and H 2 S. Dysregulation of matricellular proteins contributes to the pathogenesis of vascular diseases and cancer. Defining the molecular mechanisms and receptors involved is revealing new therapeutic opportunities. Recent Advances: Thrombospondin-1 (TSP1) regulates NO, H 2 S, and superoxide production and signaling in several cell types. The TSP1 receptor CD47 plays a central role in inhibition of NO signaling, but other TSP1 receptors also modulate redox signaling. The matricellular protein CCN1 engages some of the same receptors to regulate redox signaling, and ADAMTS1 regulates NO signaling in Marfan syndrome. In addition to mediating matricellular protein signaling, redox signaling is emerging as an important pathway that controls the expression of several matricellular proteins. Redox signaling remains unexplored for many matricellular proteins. Their interactions with multiple cellular receptors remains an obstacle to defining signaling mechanisms, but improved transgenic models could overcome this barrier. Therapeutics targeting the TSP1 receptor CD47 may have beneficial effects for treating cardiovascular disease and cancer and have recently entered clinical trials. Biomarkers are needed to assess their effects on redox signaling in patients and to evaluate how these contribute to their therapeutic efficacy and potential side effects. Antioxid. Redox Signal. 27, 874-911.

  10. Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

    DEFF Research Database (Denmark)

    Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

    2015-01-01

    In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...... inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair...

  11. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    Science.gov (United States)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  12. Dual localized mitochondrial and nuclear proteins as gene expression regulators in plants?

    Directory of Open Access Journals (Sweden)

    Philippe eGiegé

    2012-09-01

    Full Text Available Mitochondria heavily depend on the coordinated expression of both mitochondrial and nuclear genomes because some of their most significant activities are held by multi-subunit complexes composed of both mitochondrial and nuclear encoded proteins. Thus, precise communication and signaling pathways are believed to exist between the two compartments. Proteins dual localized to both mitochondria and the nucleus make excellent candidates for a potential involvement in the envisaged communication. Here, we review the identified instances of dual localized nucleo-mitochondrial proteins with an emphasis on plant proteins and discuss their functions, which are seemingly mostly related to gene expression regulation. We discuss whether dual localization could be achieved by dual targeting and / or by re-localization and try to apprehend the signals required for the respective processes. Finally, we propose that in some instances, dual localized mitochondrial and nuclear proteins might act as retrograde signaling molecules for mitochondrial biogenesis.

  13. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  14. Histone turnover within nonproliferating cells

    International Nuclear Information System (INIS)

    Commerford, S.L.; Carsten, A.L.; Cronkite, E.P.

    1982-01-01

    The turnover of DNA and histones in the livers and brains of mice has been determined. These mice had been exposed to constant levels of tritiated water from conception until they were 8 months old. At this point, exposure to tritium was discontinued, and the tritium remaining in DNA and histones was measured at various intervals afterward. The half-lives calculated for these components (with 95% confidence limits given in parentheses) were 117 (85 to 188) days for liver histone, 318 (241 to 466) days for liver DNA, 159 (129 to 208) days for brain histone and 593 (376 to 1406) days for brain DNA. The difference between histone and DNA turnover is statistically significant for both tissues and indicates that histone turnover within tissues cannot be solely accounted for by cell turnover within the tissue but also must include histone turnover within living cells. The half-life of histone within cells is estimated to be 117 (88 to 178) days in liver and 223 (187 to 277) days in brain

  15. PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78.

    Science.gov (United States)

    Moriya, Chiharu; Taniguchi, Hiroaki; Nagatoishi, Satoru; Igarashi, Hisayoshi; Tsumoto, Kouhei; Imai, Kohzoh

    2018-02-01

    PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem-like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple-negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates-glucose-regulated protein 78 (GRP78) and heat shock protein 90-α (HSP90α)-were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA-MB231). Surface plasmon resonance analysis using GST-PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C-terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase-based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem-like CD24 -  CD44 + and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  16. Uptake and degradation of circulating proteins by the liver.

    NARCIS (Netherlands)

    Buys, Carolus Henricus Cornelis Maria

    1976-01-01

    Circulating proteins, like all proteins in a living animal, are subject to continual replacement or turnover. This process implies both synthesis and degradation, This thesis deals with the degradative part of turnover of circulating proteins. ... Summary

  17. Proteomics analysis of apoptosis-regulating proteins in tissues with different radiosensitivity

    International Nuclear Information System (INIS)

    An, Jeung-Hee; Seong, Jin-Sil

    2006-01-01

    The aim of this study was to identify of radiosusceptibility proteins in tissues with different radiosensitivity. C3H/HeJ mice were exposed to 10 Gy. The tissues were processed for proteins extraction and were analyzed by 2-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionizing time-of-flight mass spectrometry and validated by immunohistochemical staining and Western blotting. The peaks of apoptosis levels were 35.3±1.7% and 0.6±0.2% in the spleen and the liver, respectively, after ionizing radiation. Analysis of liver tissue showed that the expression level of reactive oxygen species (ROS) related proteins such as cytochrome c, glutathione S transferase, NADH dehydrogenase and peroxiredoxin VI increased after radiation. The expression level of cytochrome c increased to 3-fold after ionizing radiation in both tissues. However in spleen tissue, the expression level of various kinds of apoptosis regulating proteins increased after radiation. These involved iodothyronine, CD 59A glycoprotein precursor, fas antigen and tumor necrosis factor -inducible protein TSG-6nprecursor after radiation. The difference in the apoptosis index between the liver and spleen tissues is closely associated with the expression of various kinds of apoptosis-related proteins. The result suggests that the expression of apoptosis-related protein and redox proteins play important roles in this radiosusceptibility. (author)

  18. Multiple structure-intrinsic disorder interactions regulate and coordinate Hox protein function

    Science.gov (United States)

    Bondos, Sarah

    During animal development, Hox transcription factors determine fate of developing tissues to generate diverse organs and appendages. Hox proteins are famous for their bizarre mutant phenotypes, such as replacing antennae with legs. Clearly, the functions of individual Hox proteins must be distinct and reliable in vivo, or the organism risks malformation or death. However, within the Hox protein family, the DNA-binding homeodomains are highly conserved and the amino acids that contact DNA are nearly invariant. These observations raise the question: How do different Hox proteins correctly identify their distinct target genes using a common DNA binding domain? One possible means to modulate DNA binding is through the influence of the non-homeodomain protein regions, which differ significantly among Hox proteins. However genetic approaches never detected intra-protein interactions, and early biochemical attempts were hindered because the special features of ``intrinsically disordered'' sequences were not appreciated. We propose the first-ever structural model of a Hox protein to explain how specific contacts between distant, intrinsically disordered regions of the protein and the homeodomain regulate DNA binding and coordinate this activity with other Hox molecular functions.

  19. CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

    Science.gov (United States)

    Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

    2017-06-15

    Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Effect of regional muscle location but not adiposity on mitochondrial biogenesis-regulating proteins

    DEFF Research Database (Denmark)

    Ponce-González, Jesús Gustavo; Ara, Ignacio; Larsen, Steen

    2016-01-01

    PURPOSE: The aim of this study was to determine if the expression of the mitochondrial biogenesis-regulating proteins SIRT1, SIRT3 and PGC-1alpha in human skeletal muscle is influenced by adiposity. METHOD: Twenty-nine male subjects were recruited into three groups: control (n = 10), obese (n = 10...

  1. Down-regulation of E protein activity augments an ILC2 differentiation program in the thymus

    Science.gov (United States)

    Innate lymphoid cells (ILCs) are important regulators in various immune responses. Current paradigm states that all newly-made ILCs originate from common lymphoid progenitors (CLP) in the bone marrow. Id2, an inhibitor of E protein transcription factors, is indispensable for ILC differentiation. Une...

  2. ZNF143 protein is an important regulator of the myeloid transcription factor C/EBP

    Czech Academy of Sciences Publication Activity Database

    Gonzalez, D.; Luyten, A.; Bartholdy, B.; Zhou, Q.; Kardošová, Miroslava; Ebralidze, A.; Swanson, K.D.; Radomska, H.S.; Zhang, P.; Kobayashi, S.S.; Welner, R.S.; Levantini, E.; Steidl, U.; Chong, G.; Collombet, S.; Choi, M.H.; Friedman, A.D.; Scott, L.M.; Alberich-Jorda, Meritxell; Tenen, D.G.

    2017-01-01

    Roč. 292, č. 46 (2017), s. 18924-18936 ISSN 0021-9258 Institutional support: RVO:68378050 Keywords : CCAAT-enhancer-binding protein * gene regulation * hematopoiesis * promoter * transcription factor * EBPalpha * ZNF143 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.125, year: 2016

  3. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  4. Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2

    DEFF Research Database (Denmark)

    Yang, Yaoming; Duguay, David; Bédard, Nathalie

    2012-01-01

    Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock...

  5. Gender-Dimorphic Regulation of Skeletal Muscle Proteins in Streptozotocin-Induced Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Minji Choi

    2013-03-01

    Full Text Available Background: Despite the fact that sexual differences increase diabetic risk and contribute to the need for gender-specific care, there remain contradictory results as to whether or not sexual dimorphism increases susceptibility to the development of type 1 diabetes mellitus. Methods: To examine gender-dimorphic regulation of skeletal muscle proteins between healthy control and STZ-induced diabetic rats of both genders, we performed differential proteome analysis using two-dimensional electrophoresis combined with mass spectrometry. Results: Animal experiments revealed that STZ treatment rendered female rats more susceptible to induction of diabetes than their male littermates with significantly lower plasma insulin levels due to hormonal regulation. Proteomic analysis of skeletal muscle identified a total of 21 proteins showing gender-dimorphic differential expression patterns between healthy controls and diabetic rats. Most interestingly, gender-specific proteome comparison showed that male and female rats displayed differential regulation of proteins involved in muscle contraction, carbohydrate, and lipid metabolism, as well as oxidative phosphorylation and cellular stress. Conclusion: The current proteomic study revealed that impaired protein regulation was more prominent in the muscle tissue of female diabetic rats, which were more susceptible to STZ-induced diabetes. We expect that the present proteomic data can provide valuable information for evidence-based gender-specific treatment of diabetes.

  6. Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

    Science.gov (United States)

    Chaikam, Vijay; Karlson, Dale T

    2010-01-01

    The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

  7. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Directory of Open Access Journals (Sweden)

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  8. Deleted in breast cancer 1 (DBC1) protein regulates hepatic gluconeogenesis.

    Science.gov (United States)

    Nin, Veronica; Chini, Claudia C S; Escande, Carlos; Capellini, Verena; Chini, Eduardo N

    2014-02-28

    Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

  9. Controlling parasympathetic regulation of heart rate: a gatekeeperrole for RGS proteins in the sinoatrial node

    Directory of Open Access Journals (Sweden)

    Alexandra eMighiu

    2012-06-01

    Full Text Available Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the SAN exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the sinoatrial node (SAN is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G protein-coupled inwardly-rectifying potassium channel (GIRK channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs and L-type Ca2+ channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, sinus node dysfunction, and atrial fibrillation.

  10. Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

    Science.gov (United States)

    Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

    2006-07-01

    The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

  11. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Tomoyo Ujisawa

    Full Text Available Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

  12. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

    Science.gov (United States)

    Ujisawa, Tomoyo; Ohta, Akane; Uda-Yagi, Misato

    2016-01-01

    Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron. PMID:27788246

  13. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion

    Directory of Open Access Journals (Sweden)

    Nisha Durand

    2016-02-01

    Full Text Available The Protein Kinase D (PKD isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs and diacylglycerol (DAG. PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  15. The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.

    Science.gov (United States)

    Smith, Jeffrey S; Rajagopal, Sudarshan

    2016-04-22

    The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Salinity Regulates Claudin mRNA and Protein Expression in the Teleost Gill

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Baltzegar, David A; Ozden, Ozkan

    2008-01-01

    The teleost gill carries out NaCl uptake in fresh water (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight junctional claudins during salinity...... was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer and staining appears more intense in gill of FW versus SW fish. Additionally, tilapia claudin 28a and 30 genes were characterized......, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated...

  17. Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB-1 from rice

    NARCIS (Netherlands)

    Chen, Y.; Sun, A.; Wang, M.; Zhu, Z.; Ouwerkerk, P.B.F.

    2014-01-01

    Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we

  18. Recent evolution of the NF-κB and inflammasome regulating protein POP2 in primates

    Directory of Open Access Journals (Sweden)

    Harton Jonathan A

    2011-03-01

    Full Text Available Abstract Background Pyrin-only protein 2 (POP2 is a small human protein comprised solely of a pyrin domain that inhibits NF-κB p65/RelA and blocks the formation of functional IL-1β processing inflammasomes. Pyrin proteins are abundant in mammals and several, like POP2, have been linked to activation or regulation of inflammatory processes. Because POP2 knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether POP2 is common in the mammalian lineage. Results BLAST searches revealed that POP2 is absent from the available genomes of not only mice and rats, but those of other domestic mammals and New World monkeys as well. POP2 is however present in the genome of the primate species most closely related to humans including Pan troglodytes (chimpanzees, Macaca mulatta (rhesus macaques and others. Interestingly, chimpanzee POP2 is identical to human POP2 (huPOP2 at both the DNA and protein level. Macaque POP2 (mqPOP2, although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. Further, POP2 appears to have arisen in the mammalian genome relatively recently (~25 mya and likely derived from retrogene insertion of NLRP2. Conclusion Our findings support the hypothesis that the NLR loci of mammals, encoding proteins involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures. Since POP2 is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and Old World primates further suggests that additional regulation of these signals is important in these species.

  19. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

    Directory of Open Access Journals (Sweden)

    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

  20. Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

    KAUST Repository

    Skinner, John J.

    2017-12-05

    Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

  1. Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

    KAUST Repository

    Skinner, John J.; Wang, Sheng; Lee, Jiyoung; Ong, Colin; Sommese, Ruth; Sivaramakrishnan, Sivaraj; Koelmel, Wolfgang; Hirschbeck, Maria; Schindelin, Hermann; Kisker, Caroline; Lorenz, Kristina; Sosnick, Tobin R.; Rosner, Marsha Rich

    2017-01-01

    Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

  2. Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation.

    Science.gov (United States)

    Lipton, Stuart A; Choi, Yun-Beom; Takahashi, Hiroto; Zhang, Dongxian; Li, Weizhong; Godzik, Adam; Bankston, Laurie A

    2002-09-01

    Until recently cysteine residues, especially those located extracellularly, were thought to be important for metal coordination, catalysis and protein structure by forming disulfide bonds - but they were not thought to regulate protein function. However, this is not the case. Crucial cysteine residues can be involved in modulation of protein activity and signaling events via other reactions of their thiol (sulfhydryl; -SH) groups. These reactions can take several forms, such as redox events (chemical reduction or oxidation), chelation of transition metals (chiefly Zn(2+), Mn(2+) and Cu(2+)) or S-nitrosylation [the catalyzed transfer of a nitric oxide (NO) group to a thiol group]. In several cases, these disparate reactions can compete with one another for the same thiol group on a single cysteine residue, forming a molecular switch composed of a latticework of possible redox, NO or Zn(2+) modifications to control protein function. Thiol-mediated regulation of protein function can also involve reactions of cysteine residues that affect ligand binding allosterically. This article reviews the basis for these molecular cysteine switches, drawing on the NMDA receptor as an exemplary protein, and proposes a molecular model for the action of S-nitrosylation based on recently derived crystal structures.

  3. Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes.

    Science.gov (United States)

    Sawaki, Yoshiharu; Kobayashi, Yuriko; Kihara-Doi, Tomonori; Nishikubo, Nobuyuki; Kawazu, Tetsu; Kobayashi, Masatomo; Kobayashi, Yasufumi; Iuchi, Satoshi; Koyama, Hiroyuki; Sato, Shigeru

    2014-06-01

    Tolerance to soil acidity is an important trait for eucalyptus clones that are introduced to commercial forestry plantations in pacific Asian countries, where acidic soil is dominant in many locations. A conserved transcription factor regulating aluminum (Al) and proton (H⁺) tolerance in land-plant species, STOP1 (SENSITIVE TOPROTON RHIZOTOXICITY 1)-like protein, was isolated by polymerase chain reaction-based cloning, and then suppressed by RNA interference in hairy roots produced by Agrobacterium rhizogenes-mediated transformation. Eucalyptus STOP1-like protein complemented proton tolerance in an Arabidopsis thaliana stop1-mutant, and localized to the nucleus in a transient assay of a green fluorescent protein fusion protein expressed in tobacco leaves by Agrobacterium tumefaciens-mediated transformation. Genes encoding a citrate transporting MULTIDRUGS AND TOXIC COMPOUND EXTRUSION protein and an orthologue of ALUMINUM SENSITIVE 3 were suppressed in transgenic hairy roots in which the STOP1 orthologue was knocked down. In summary, we identified a series of genes for Al-tolerance in eucalyptus, including a gene for STOP1-like protein and the Al-tolerance genes it regulates. These genes may be useful for molecular breeding and genomic selection of elite clones to introduce into acid soil regions. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-02-01

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

  5. Job turnover and labor turnover : a taxonomy of employment dynamics

    NARCIS (Netherlands)

    Hassink, Wolter H.J.; Hamermesh, Daniel S.

    1994-01-01

    We present an organized set of stylized facts on the relations among flows of workers,changes in employment and changes in the numer of jobs at the firm level. Job turnover isusually measured by comparing stocks of employment in each firm at two points in time andadding up the absolute employment

  6. Versatile function of the circadian protein CIPC as a regulator of Erk activation

    International Nuclear Information System (INIS)

    Matsunaga, Ryota; Nishino, Tasuku; Yokoyama, Atsushi; Nakashima, Akio; Kikkawa, Ushio; Konishi, Hiroaki

    2016-01-01

    The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.

  7. Versatile function of the circadian protein CIPC as a regulator of Erk activation

    Energy Technology Data Exchange (ETDEWEB)

    Matsunaga, Ryota; Nishino, Tasuku [Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023 (Japan); Yokoyama, Atsushi [Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Nakashima, Akio; Kikkawa, Ushio [Biosignal Research Center, Kobe University, Kobe 657-8501 (Japan); Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp [Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023 (Japan)

    2016-01-15

    The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.

  8. Regulation of the vertebrate cell cycle by the cdc2 protein kinase

    International Nuclear Information System (INIS)

    Draetta, G.; Brizuela, L.; Moran, B.; Beach, D.

    1988-01-01

    A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. The authors have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. They find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G 1 but displays maximal enzymatic activity during mitotic metaphase. These observations indicate that in a wide variety of vertebrate cells, the cdc2 protein kinase is involved in regulating mitosis. The authors' approach taken toward study of the cdc2 protein kinase highlights the possibilities that now exist for combining the advantages of ascomycete genetics with the cell-free systems of Xenopus and the biochemical advantages of tissue culture cells to investigate fundamental problems of the cell cycle

  9. Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

    Science.gov (United States)

    Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

    2018-04-30

    Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. One, Two, Three: Polycomb Proteins Hit All Dimensions of Gene Regulation

    Directory of Open Access Journals (Sweden)

    Stefania del Prete

    2015-07-01

    Full Text Available Polycomb group (PcG proteins contribute to the formation and maintenance of a specific repressive chromatin state that prevents the expression of genes in a particular space and time. Polycomb repressive complexes (PRCs consist of several PcG proteins with specific regulatory or catalytic properties. PRCs are recruited to thousands of target genes, and various recruitment factors, including DNA-binding proteins and non-coding RNAs, are involved in the targeting. PcG proteins contribute to a multitude of biological processes by altering chromatin features at different scales. PcG proteins mediate both biochemical modifications of histone tails and biophysical modifications (e.g., chromatin fiber compaction and three-dimensional (3D chromatin conformation. Here, we review the role of PcG proteins in nuclear architecture, describing their impact on the structure of the chromatin fiber, on chromatin interactions, and on the spatial organization of the genome in nuclei. Although little is known about the role of plant PcG proteins in nuclear organization, much is known in the animal field, and we highlight similarities and differences in the roles of PcG proteins in 3D gene regulation in plants and animals.

  11. Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

    Science.gov (United States)

    Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

    2007-02-01

    The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

  12. Huntingtin-interacting protein 14 is a type 1 diabetes candidate protein regulating insulin secretion and β-cell apoptosis

    DEFF Research Database (Denmark)

    Berchtold, Lukas Adrian; Størling, Zenia Marian; Ortis, Fernanda

    2011-01-01

    Type 1 diabetes (T1D) is a complex disease characterized by the loss of insulin-secreting β-cells. Although the disease has a strong genetic component, and several loci are known to increase T1D susceptibility risk, only few causal genes have currently been identified. To identify disease...... genes in T1D, including the INS gene. An unexpected top-scoring candidate gene was huntingtin-interacting protein (HIP)-14/ZDHHC17. Immunohistochemical analysis of pancreatic sections demonstrated that HIP14 is almost exclusively expressed in insulin-positive cells in islets of Langerhans. RNAi...... knockdown experiments established that HIP14 is an antiapoptotic protein required for β-cell survival and glucose-stimulated insulin secretion. Proinflammatory cytokines (IL-1β and IFN-γ) that mediate β-cell dysfunction in T1D down-regulated HIP14 expression in insulin-secreting INS-1 cells and in isolated...

  13. Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System

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    Kexin Zhao

    2017-05-01

    Full Text Available Lipoprotein cholesterol is delivered to the limiting membrane of late endosomes/lysosomes (LELs by Niemann-Pick C1 (NPC1. However, the mechanism of cholesterol transport from LELs to the endoplasmic reticulum (ER is poorly characterized. We report that oxysterol-binding protein-related protein 1L (ORP1L is necessary for this stage of cholesterol export. CRISPR-mediated knockout of ORP1L in HeLa and HEK293 cells reduced esterification of cholesterol to the level in NPC1 knockout cells, and it increased the expression of sterol-regulated genes and de novo cholesterol synthesis, indicative of a block in cholesterol transport to the ER. In the absence of this transport pathway, cholesterol-enriched LELs accumulated in the Golgi/perinuclear region. Cholesterol delivery to the ER required the sterol-, phosphatidylinositol 4-phosphate-, and vesicle-associated membrane protein-associated protein (VAP-binding activities of ORP1L, as well as NPC1 expression. These results suggest that ORP1L-dependent membrane contacts between LELs and the ER coordinate cholesterol transfer with the retrograde movement of endo-lysosomal vesicles.

  14. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

    2006-01-01

    Microbial biofilm formation often causes problems in medical and industrial settings, and knowledge about the factors that are involved in biofilm development and dispersion is useful for creating strategies to control the processes. In this report, we present evidence that proteins with GGDEF...... and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli Yhj......H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

  15. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    Science.gov (United States)

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  16. SET protein up-regulated testosterone production in the cultured preantral follicles

    Directory of Open Access Journals (Sweden)

    Xu Boqun

    2013-02-01

    Full Text Available Abstract Background We found previously that the expression of SET gene was up-regulated in polycystic ovaries. Evidences suggested that SET protein was essential for regulating both the promoter activity of CYP17A1 and the biological activity of P450c17. In this study, we explored whether SET regulated androgen production in preantral follicles. Methods The mouse preantral follicles were cultured in vitro. Testosterone secretion and expression of steroidogenic enzymes were observed in the preantral follicles treated in vitro by SET overexpression and knockdown. Results Testosterone levels in the media of the AdCMV-SET infected follicles significantly increased, and the CYP17A1 and HSD3B2 expression also significantly increased (P P  Conclusions SET played a positive role in regulating ovarian androgen biosynthesis by enhancing the transcription of steroidogenic enzymes CYP17A1 and HSD3B2, which maybe contribute to the hyperandrogenism in PCOS.

  17. Health insurance, cost expectations, and adverse job turnover.

    Science.gov (United States)

    Ellis, Randall P; Albert Ma, Ching-To

    2011-01-01

    Because less healthy employees value health insurance more than the healthy ones, when health insurance is newly offered job turnover rates for healthier employees decline less than turnover rates for the less healthy. We call this adverse job turnover, and it implies that a firm's expected health costs will increase when health insurance is first offered. Health insurance premiums may fail to adjust sufficiently fast because state regulations restrict annual premium changes, or insurers are reluctant to change premiums rapidly. Even with premiums set at the long run expected costs, some firms may be charged premiums higher than their current expected costs and choose not to offer insurance. High administrative costs at small firms exacerbate this dynamic selection problem. Using 1998-1999 MEDSTAT MarketScan and 1997 Employer Health Insurance Survey data, we find that expected employee health expenditures at firms that offer insurance have lower within-firm and higher between-firm variance than at firms that do not. Turnover rates are systematically higher in industries in which firms are less likely to offer insurance. Simulations of the offer decision capturing between-firm health-cost heterogeneity and expected turnover rates match the observed pattern across firm sizes well. 2010 John Wiley & Sons, Ltd.

  18. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  19. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    International Nuclear Information System (INIS)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-01-01

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

  20. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    Science.gov (United States)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

  1. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  2. An SMC-like protein binds and regulates Caenorhabditis elegans condensins.

    Directory of Open Access Journals (Sweden)

    Lucy Fang-I Chao

    2017-03-01

    Full Text Available Structural Maintenance of Chromosomes (SMC family proteins participate in multisubunit complexes that govern chromosome structure and dynamics. SMC-containing condensin complexes create chromosome topologies essential for mitosis/meiosis, gene expression, recombination, and repair. Many eukaryotes have two condensin complexes (I and II; C. elegans has three (I, II, and the X-chromosome specialized condensin IDC and their regulation is poorly understood. Here we identify a novel SMC-like protein, SMCL-1, that binds to C. elegans condensin SMC subunits, and modulates condensin functions. Consistent with a possible role as a negative regulator, loss of SMCL-1 partially rescued the lethal and sterile phenotypes of a hypomorphic condensin mutant, while over-expression of SMCL-1 caused lethality, chromosome mis-segregation, and disruption of condensin IDC localization on X chromosomes. Unlike canonical SMC proteins, SMCL-1 lacks hinge and coil domains, and its ATPase domain lacks conserved amino acids required for ATP hydrolysis, leading to the speculation that it may inhibit condensin ATPase activity. SMCL-1 homologs are apparent only in the subset of Caenorhabditis species in which the condensin I and II subunit SMC-4 duplicated to create the condensin IDC- specific subunit DPY-27, suggesting that SMCL-1 helps this lineage cope with the regulatory challenges imposed by evolution of a third condensin complex. Our findings uncover a new regulator of condensins and highlight how the duplication and divergence of SMC complex components in various lineages has created new proteins with diverse functions in chromosome dynamics.

  3. Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

    Directory of Open Access Journals (Sweden)

    Sven I Walaas

    2011-08-01

    Full Text Available Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington's disease and Parkinson's disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly DARPP-32, RCS (Regulator of Calmodulin Signaling and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways.

  4. Rho proteins − the key regulators of cytoskeleton in the progression of mitosis and cytokinesis

    Directory of Open Access Journals (Sweden)

    Anna Klimaszewska

    2011-11-01

    Full Text Available The Rho proteins are members of the Ras superfamily of small GTPases. They are thought to be crucial regulators of multiple signal transduction pathways that influence a wide range of cellular functions, including migration, membrane trafficking, adhesion, polarity and cell shape changes. Thanks to their ability to control the assembly and organization of the actin and microtubule cytoskeletons, Rho GTPases are known to regulate mitosis and cytokinesis progression. These proteins are required for formation and rigidity of the cortex during mitotic cell rounding, mitotic spindle formation and attachment of the spindle microtubules to the kinetochore. In addition, during cytokinesis, they are involved in promoting division plane determination, contractile ring and cleavage furrow formation and abscission. They are also known as regulators of cell cycle progression at the G1/S and G2/M transition. Thus, the signal transduction pathways in which Rho proteins participate, appear to connect dynamics of actin and microtubule cytoskeletons to cell cycle progression. We review the current state of knowledge concerning the molecular mechanisms by which Rho GTPase signaling regulates remodeling of actin and microtubule cytoskeletons in order to control cell division progression.

  5. Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yuping Luo

    2010-04-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP. FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs. We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

  6. Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

    Directory of Open Access Journals (Sweden)

    Honghao Li

    Full Text Available Although intracellular beta amyloid (Aβ accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP deposition including unfolded protein response (UPR, ubiquitin proteasome system (UPS activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94, glucose-regulated protein 78 (GRP78, calreticulin and calnexin and valosin containing protein (VCP were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

  7. A role for PERK in the mechanism underlying fluoride-induced bone turnover

    International Nuclear Information System (INIS)

    Sun, Fei; Li, Xining; Yang, Chen; Lv, Peng; Li, Guangsheng; Xu, Hui

    2014-01-01

    While it has been well-documented that excessive fluoride exposure caused the skeletal disease and osteoblasts played a critical role in the advanced skeletal fluorosis, the underlying mechanism that mediated these effects remain poorly understood. The present study was undertaken to examine the effect of fluoride on bone of rats and MC3T3-E1 cells in vitro. Herein we found pathological features of high bone turnover in fluoride-treated rats, which was supported by an increase of osteogenic and osteoclastogenic genes expression in different stages of fluoride exposure. The skeletal toxicity of fluoride was accompanied by activation of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). A novel finding of this study was that expression of PKR-like endoplasmic reticulum kinase (PERK) was the same trend with receptor activator for nuclear factor-κ B ligand (RANKL), and NF-E2 p45-related factor 2 (Nrf2) was the same trend with Runt-related transcription factor 2 (Runx2) in bones of rats exposed to varied fluoride condition. Based on these data, we hypothesized that up-regulation of PERK probably played a role in mediating bone turnover induced by fluoride. Action of fluoride on MC3T3-E1 cells differentiation was demonstrated through analysis of alkaline phosphatase (ALP) activity and mineralized nodules formation. Meantime, an increase of binding immunoglobulin protein (BiP) expression indicated the active ER stress in cells exposed to various dose of fluoride. Blocking PERK expression using siRNA showed the obvious decrease of osteogenic and osteoclastogenic factors expression in MC3T3-E1 cells exposed to certain dose of fluoride that could positively stimulate osteoblastic viability. In conclusion these findings underscore the importance of PERK in modulating fluoride induced bone formation and bone resorption. Understanding the link between PERK and bone turnover could probe into the mechanism underlying different bone lesion of

  8. The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

    Science.gov (United States)

    Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

    2017-10-13

    Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Employee turnover: measuring the malady.

    Science.gov (United States)

    O'Connor, Stephen

    2002-01-01

    One measure of an organization's value to its employees is turnover. But how do you know if your employees are wondering if the grass is greener elsewhere? Scott Badler in his book What's So Funny about Looking for a Job? suggests a quick quiz to find out.

  10. Biochemical markers of bone turnover

    International Nuclear Information System (INIS)

    Kim, Deog Yoon

    1999-01-01

    Biochemical markers of bone turnover has received increasing attention over the past few years, because of the need for sensitivity and specific tool in the clinical investigation of osteoporosis. Bone markers should be unique to bone, reflect changes of bone less, and should be correlated with radiocalcium kinetics, histomorphometry, or changes in bone mass. The markers also should be useful in monitoring treatment efficacy. Although no bone marker has been established to meet all these criteria, currently osteocalcin and pyridinium crosslinks are the most efficient markers to assess the level of bone turnover in the menopausal and senile osteoporosis. Recently, N-terminal telopeptide (NTX), C-terminal telopeptide (CTX) and bone specific alkaline phosphatase are considered as new valid markers of bone turnover. Recent data suggest that CTX and free deoxypyridinoline could predict the subsequent risk of hip fracture of elderly women. Treatment of postmenopausal women with estrogen, calcitonin and bisphosphonates demonstrated rapid decrease of the levels of bone markers that correlated with the long-term increase of bone mass. Factors such as circadian rhythms, diet, age, sex, bone mass and renal function affect the results of biochemical markers and should be appropriately adjusted whenever possible. Each biochemical markers of bone turnover may have its own specific advantages and limitations. Recent advances in research will provide more sensitive and specific assays

  11. Phosphorylation Regulates the Bound Structure of an Intrinsically Disordered Protein: The p53-TAZ2 Case.

    Directory of Open Access Journals (Sweden)

    Raúl Esteban Ithuralde

    Full Text Available Disordered regions and Intrinsically Disordered Proteins (IDPs are involved in critical cellular processes and may acquire a stable three-dimensional structure only upon binding to their partners. IDPs may follow a folding-after-binding process, known as induced folding, or a folding-before-binding process, known as conformational selection. The transcription factor p53 is involved in the regulation of cellular events that arise upon stress or DNA damage. The p53 domain structure is composed of an N-terminal transactivation domain (p53TAD, a DNA Binding Domain and a tetramerization domain. The activity of TAD is tightly regulated by interactions with cofactors, inhibitors and phosphorylation. To initiate transcription, p53TAD binds to the TAZ2 domain of CBP, a co-transcription factor, and undergoes a folding and binding process, as revealed by the recent NMR structure of the complex. The activity of p53 is regulated by phosphorylation at multiple sites on the TAD domain and recent studies have shown that modifications at three residues affect the binding towards TAZ2. However, we still do not know how these phosphorylations affect the structure of the bound state and, therefore, how they regulate the p53 function. In this work, we have used computational simulations to understand how phosphorylation affects the structure of the p53TAD:TAZ2 complex and regulates the recognition mechanism. Phosphorylation has been proposed to enhance binding by direct interaction with the folded protein or by changing the unbound conformation of IDPs, for example by pre-folding the protein favoring the recognition mechanism. Here, we show an interesting turn in the p53 case: phosphorylation mainly affects the bound structure of p53TAD, highlighting the complexity of IDP protein-protein interactions. Our results are in agreement with previous experimental studies, allowing a clear picture of how p53 is regulated by phosphorylation and giving new insights into how

  12. Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis.

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

    Full Text Available Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa, cells on soft substrates (150-300 Pa exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins and glycolysis (e.g., phosphofructokinase-1, whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical

  13. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase.

    Directory of Open Access Journals (Sweden)

    Kara R Barber

    2017-02-01

    Full Text Available Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated.

  14. Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders

    Directory of Open Access Journals (Sweden)

    Derek W. Stouth

    2017-11-01

    Full Text Available Protein arginine methyltransferases (PRMTs are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD. PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD, spinal muscular atrophy (SMA, and amyotrophic lateral sclerosis (ALS suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs. This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research.

  15. Lhc proteins and the regulation of photosynthetic light harvesting function by xanthophylls.

    Science.gov (United States)

    Bassi, R; Caffarri, S

    2000-01-01

    Photoprotection of the chloroplast is an important component of abiotic stress resistance in plants. Carotenoids have a central role in photoprotection. We review here the recent evidence, derived mainly from in vitro reconstitution of recombinant Lhc proteins with different carotenoids and from carotenoid biosynthesis mutants, for the existence of different mechanisms of photoprotection and regulation based on xanthophyll binding to Lhc proteins into multiple sites and the exchange of chromophores between different Lhc proteins during exposure of plants to high light stress and the operation of the xanthophyll cycle. The use of recombinant Lhc proteins has revealed up to four binding sites in members of Lhc families with distinct selectivity for xanthophyll species which are here hypothesised to have different functions. Site L1 is selective for lutein and is here proposed to be essential for catalysing the protection from singlet oxygen by quenching chlorophyll triplets. Site L2 and N1 are here proposed to act as allosteric sites involved in the regulation of chlorophyll singlet excited states by exchanging ligand during the operation of the xanthophyll cycle. Site V1 of the major antenna complex LHC II is here hypothesised to be a deposit for readily available substrate for violaxanthin de-epoxidase rather than a light harvesting pigment. Moreover, xanthophylls bound to Lhc proteins can be released into the lipid bilayer where they contribute to the scavenging of reactive oxygen species produced in excess light.

  16. Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

    Science.gov (United States)

    Khan, Sheraz; Iqbal, Mazhar; Tariq, Muhammad; Baig, Shahid M; Abbas, Wasim

    2018-01-01

    HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

  17. Regulation of multispanning membrane protein topology via post-translational annealing.

    Science.gov (United States)

    Van Lehn, Reid C; Zhang, Bin; Miller, Thomas F

    2015-09-26

    The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

  18. WRKY proteins: signaling and regulation of expression during abiotic stress responses.

    Science.gov (United States)

    Banerjee, Aditya; Roychoudhury, Aryadeep

    2015-01-01

    WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research.

  19. Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age- and sex-dependent remodeling of the extracellular matrix.

    Science.gov (United States)

    Kehlet, Stephanie N; Willumsen, Nicholas; Armbrecht, Gabriele; Dietzel, Roswitha; Brix, Susanne; Henriksen, Kim; Karsdal, Morten A

    2018-01-01

    The extracellular matrix (ECM) plays a vital role in maintaining normal tissue function. Collagens are major components of the ECM and there is a tight equilibrium between degradation and formation of these proteins ensuring tissue health and homeostasis. As a consequence of tissue turnover, small collagen fragments are released into the circulation, which act as important biomarkers in the study of certain tissue-related remodeling factors in health and disease. The aim of this study was to establish an age-related collagen turnover profile of the main collagens of the interstitial matrix (type I and III collagen) and basement membrane (type IV collagen) in healthy men and women. By using well-characterized competitive ELISA-assays, we assessed specific fragments of degraded (C1M, C3M, C4M) and formed (PINP, Pro-C3, P4NP7S) type I, III and IV collagen in serum from 617 healthy men and women ranging in ages from 22 to 86. Subjects were divided into 5-year age groups according to their sex and age. Groups were compared using Kruskal-Wallis adjusted for Dunn's multiple comparisons test and Mann-Whitney t-test. Age-specific changes in collagen turnover was most profound for type I collagen. PINP levels decreased in men with advancing age, whereas in women, the level decreased in early adulthood followed by an increase around the age of menopause (age 40-60). Sex-specific changes in type I, III and IV collagen turnover was present at the age around menopause (age 40-60) with women having an increased turnover. In summary, collagen turnover is affected by age and sex with the interstitial matrix and the basement membrane being differently regulated. The observed changes needs to be accounted for when measuring ECM related biomarkers in clinical studies.

  20. Whole-body protein turnover and energy expenditure in post-viral hepatocirrhotic patients. A study using multiple stable isotope tracers to estimate protein and energy requirements and the efficacy of a new diet therapy based on Chinese food

    International Nuclear Information System (INIS)

    Xia, Z.Q.; Dai, T.C.; Luo, W.

    1993-01-01

    L-[1- 13 C]-leucine and 15 N-glycine doubly-labelled tracer experiments revealed accelerated kinetics of leucine, glycine and whole-body protein in post-viral hepatocirrhotic patients. Together with the results of nitrogen balance measurement, the daily protein requirement of these patients was estimated to be higher than 1.2 g/kg/d. Doubly labelled water experiments and NaH 13 CO 3 experiments revealed that the freely living and basal energy expenditure of post-viral hepatocirrhotic patients was not different from that in normal subjects with comparable physical and mental activities. For those freely living in hospital, the energy requirements is estimated to be 150-160 kJ/kg/d. According to the above results, a therapeutic diet formulation based on Chinese food was designed for the patients which contained 1.5 g/kg/d of protein and 150-160 kJ/kg/d. 60-70% of the dietary protein was of vegetable origin, with a branched chain amino acid/aromatic amino acid ratio slightly but significantly higher than the common hospital diet. Patients with compensated post-viral hepatocirrhosis adapted to the diet rapidly. After two months' therapy, the negative nitrogen balance turned positive along with an increase of body weight and urinary creatinine, indicating and improvement of general nutritional status, probably with accumulation of muscle protein. The diet is relatively cheap, can be easily handled by the patients themselves, and hence is also applicable to outpatients. 54 refs, 8 tabs

  1. Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer.

    Science.gov (United States)

    Michie, Alison M; McCaig, Alison M; Nakagawa, Rinako; Vukovic, Milica

    2010-01-01

    Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

  2. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  3. Dynamics of Adipocyte Turnover in Humans

    Energy Technology Data Exchange (ETDEWEB)

    Spalding, K; Arner, E; Westermark, P; Bernard, S; Buchholz, B; Bergmann, O; Blomqvist, L; Hoffstedt, J; Naslund, E; Britton, T; Concha, H; Hassan, M; Ryden, M; Frisen, J; Arner, P

    2007-07-16

    Obesity is increasing in an epidemic fashion in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for