WorldWideScience

Sample records for pseudoresponse regulator protein

  1. Coincident light and clock regulation of pseudoresponse regulator protein 37 (PRR37) controls photoperiodic flowering in sorghum

    Science.gov (United States)

    Variation in flowering time was essential during widespread crop domestication and optimal timing of reproduction remains critical to modern agriculture. Ma1, the major repressor of flowering in sorghum in long days, was identified as the pseudo-response regulator protein PRR37. Three prr37 allele...

  2. 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.

  3. A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator.

    Science.gov (United States)

    Patharkar, O R; Cushman, J C

    2000-12-01

    McCDPK1 is a salinity- and drought-induced calcium-dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum. A yeast two-hybrid experiment was performed, using full-length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins. A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1). CSP1 interacted with McCDPK1 in a substrate-like fashion in both yeast two-hybrid assays and wheat germ interaction assays. Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium-dependent manner. Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two-hybrid system can accelerate the discovery of CDPK substrates. The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudo-response regulator-like proteins that have a highly conserved helix-loop-helix DNA binding domain and a C-terminal activation domain. McCDPK1 and CSP1 co-localized to nuclei of NaCl-stressed ice plants. Csp1 transcript accumulation was not regulated by NaCl or dehydration stress. Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation.

  4. Circadian waves of expression of the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana: insight into the plant circadian clock.

    Science.gov (United States)

    Matsushika, A; Makino, S; Kojima, M; Mizuno, T

    2000-09-01

    The Arabidopsis pseudo-response regulator, APRR1, has a unique structural design containing a pseudo-receiver domain and a C-terminal CONSTANS motif. This protein was originally characterized as a presumed component of the His-to-Asp phosphorelay systems in Arabidopsis thaliana. Recently, it was reported that APRR1 is identical to the TOC1 gene product, a mutational lesion of which affects the periods of many circadian rhythms in Arabidopsis plants. TOC1 is believed to be a component of the presumed circadian clock (or central oscillator). Based on these facts, in this study four more genes, each encoding a member of the APRR1/TOC1 family of pseudo-response regulators were identified and characterized with special reference to circadian rhythms. It was found that all these members of the APRR1/TOC1 family (APRR1, APRR3, APRR5, APRR7, and APRR9) are subjected to a circadian rhythm at the level of transcription. Furthermore, in a given 24 h period, the APRR-mRNAs started accumulating sequentially after dawn with 2-3 h intervals in the order of APRR9-->APRR7-->APRR5-->APRR3-->APRR1. These sequential events of transcription, termed 'circadian waves of APRR1/TOCI', were not significantly affected by the photoperiod conditions, if any (e.g. both long and short days), and the expression of APRR9 was first boosted always after dawn. Among these APRRs, in fact, only the expression of APRR9 was rapidly and transiently induced also by white light, whereas such light responses of others were very dull, if any. These results collectively support the view that these members of the APRR1/TOC1 family are together all involved in an as yet unknown mechanism underlying the Arabidopsis circadian clock. Here we propose that the circadian waves of the APRR1/TOC1 family members are most likely a molecular basis of such a biological clock in higher plants.

  5. Comparative mapping, genomic structure, and expression analysis of eight pseudo-response regulator genes in Brassica rapa.

    Science.gov (United States)

    Kim, Jin A; Kim, Jung Sun; Hong, Joon Ki; Lee, Yeon-Hee; Choi, Beom-Soon; Seol, Young-Joo; Jeon, Chang Hoo

    2012-05-01

    Circadian clocks regulate plant growth and development in response to environmental factors. In this function, clocks influence the adaptation of species to changes in location or climate. Circadian-clock genes have been subject of intense study in models such as Arabidopsis thaliana but the results may not necessarily reflect clock functions in species with polyploid genomes, such as Brassica species, that include multiple copies of clock-related genes. The triplicate genome of Brassica rapa retains high sequence-level co-linearity with Arabidopsis genomes. In B. rapa we had previously identified five orthologs of the five known Arabidopsis pseudo-response regulator (PRR) genes that are key regulators of the circadian clock in this species. Three of these B. rapa genes, BrPRR1, BrPPR5, and BrPPR7, are present in two copies each in the B. rapa genome, for a total of eight B. rapa PRR (BrPRR) orthologs. We have now determined sequences and expression characteristics of the eight BrPRR genes and mapped their positions in the B. rapa genome. Although both members of each paralogous pair exhibited the same expression pattern, some variation in their gene structures was apparent. The BrPRR genes are tightly linked to several flowering genes. The knowledge about genome location, copy number variation and structural diversity of these B. rapa clock genes will improve our understanding of clock-related functions in this important crop. This will facilitate the development of Brassica crops for optimal growth in new environments and under changing conditions.

  6. Phylogenetic footprint of the plant clock system in angiosperms: evolutionary processes of Pseudo-Response Regulators

    Directory of Open Access Journals (Sweden)

    Saito Shigeru

    2010-05-01

    Full Text Available Abstract Background Plant circadian clocks regulate many photoperiodic and diurnal responses that are conserved among plant species. The plant circadian clock system has been uncovered in the model plant, Arabidopsis thaliana, using genetics and systems biology approaches. However, it is still not clear how the clock system had been organized in the evolutionary history of plants. We recently revealed the molecular phylogeny of LHY/CCA1 genes, one of the essential components of the clock system. The aims of this study are to reconstruct the phylogenetic relationships of angiosperm clock-associated PRR genes, the partner of the LHY/CCA1 genes, and to clarify the evolutionary history of the plant clock system in angiosperm lineages. Results In the present study, to investigate the molecular phylogeny of PRR genes, we performed two approaches: reconstruction of phylogenetic trees and examination of syntenic relationships. Phylogenetic analyses revealed that PRR genes had diverged into three clades prior to the speciation of monocots and eudicots. Furthermore, copy numbers of PRR genes have been independently increased in monocots and eudicots as a result of ancient chromosomal duplication events. Conclusions Based on the molecular phylogenies of both PRR genes and LHY/CCA1 genes, we inferred the evolutionary process of the plant clock system in angiosperms. This scenario provides evolutionary information that a common ancestor of monocots and eudicots had retained the basic components required for reconstructing a clock system and that the plant circadian clock may have become a more elaborate mechanism after the speciation of monocots and eudicots because of the gene expansion that resulted from polyploidy events.

  7. 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 ...

  8. Protein synthesis regulation by leucine

    Directory of Open Access Journals (Sweden)

    Daiana Vianna

    2010-03-01

    Full Text Available In vivo and in vitro studies have demonstrated that high protein diets affect both protein synthesis and regulation of several cellular processes. The role of amino acids as substrate for protein synthesis has been established in the literature. However, the mechanism by which these amino acids modulate transcription and regulate the mRNA translation via mTOR-dependent signaling pathway has yet to be fully determined. It has been verified that mTOR is a protein responsible for activating a cascade of biochemical intracellular events which result in the activation of the protein translation process. Of the aminoacids, leucine is the most effective in stimulating protein synthesis and reducing proteolysis. Therefore, it promotes a positive nitrogen balance, possibly by favoring the activation of this protein. This amino acid also directly and indirectly stimulates the synthesis and secretion of insulin, enhancing its anabolic cellular effects. Therefore, this review aimed to identify the role of leucine in protein synthesis modulation and to discuss the metabolic aspects related to this aminoacid.Estudos in vivo e in vitro verificaram que dietas hiperprotéicas influenciam a síntese protéica e regulam vários processos celulares. O papel dos aminoácidos como substrato para a síntese de proteínas já está bem evidenciado na literatura, porém as formas como esses aminoácidos modulam a etapa da transcrição e regulam a tradução do RNAm, pela via de sinalização dependente da mTOR, ainda não estão totalmente esclarecidas. Tem-se verificado que a mTOR é uma proteína responsável por ativar uma cascata de eventos bioquímicos intracelulares que culminam na ativação do processo de tradução protéica. Dentre todos os aminoácidos, a leucina é a mais eficaz em estimular a síntese protéica, reduzir a proteólise e, portanto, favorecer o balanço nitrogenado positivo, possivelmente por favorecer a ativação desta proteína. Al

  9. Physicochemical mechanisms of protein regulation by phosphorylation

    OpenAIRE

    Nishi, Hafumi; Shaytan, Alexey; Panchenko, Anna R.

    2014-01-01

    Phosphorylation offers a dynamic way to regulate protein activity and subcellular localization, which is achieved through reversibility and fast kinetics of posttranslational modifications. Adding or removing a dianionic phosphate group somewhere on a protein often changes the protein’s structural properties, its stability and dynamics. Moreover, the majority of signaling pathways involve an extensive set of protein-protein interactions, and phosphorylation can be used to regulate and modulat...

  10. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.

    Science.gov (United States)

    Lee, Young-Ho; Stallcup, Michael R

    2009-04-01

    Endocrine regulation frequently culminates in altered transcription of specific genes. The signal transduction pathways, which transmit the endocrine signal from cell surface to the transcription machinery, often involve posttranslational modifications of proteins. Although phosphorylation has been by far the most widely studied protein modification, recent studies have indicated important roles for other types of modification, including protein arginine methylation. Ten different protein arginine methyltransferase (PRMT) family members have been identified in mammalian cells, and numerous substrates are being identified for these PRMTs. Whereas major attention has been focused on the methylation of histones and its role in chromatin remodeling and transcriptional regulation, there are many nonhistone substrates methylated by PRMTs. This review primarily focuses on recent progress on the roles of the nonhistone protein methylation in transcription. Protein methylation of coactivators, transcription factors, and signal transducers, among other proteins, plays important roles in transcriptional regulation. Protein methylation may affect protein-protein interaction, protein-DNA or protein-RNA interaction, protein stability, subcellular localization, or enzymatic activity. Thus, protein arginine methylation is critical for regulation of transcription and potentially for various physiological/pathological processes.

  11. Regulation of protein function by ‘microProteins'

    OpenAIRE

    Staudt, Annica-Carolin; Wenkel, Stephan

    2010-01-01

    Elegant post-translational regulation is achieved by ‘microProteins', which form homotypic dimers with their targets and act through the dominant–negative suppression of protein complex function. The recent identification of new microProteins suggests their role is general and has evolved in both the plant and animal kingdoms.

  12. Physicochemical mechanisms of protein regulation by phosphorylation

    Directory of Open Access Journals (Sweden)

    Hafumi eNishi

    2014-08-01

    Full Text Available Phosphorylation offers a dynamic way to regulate protein activity and subcellular localization, which is achieved through reversibility and fast kinetics of posttranslational modifications. Adding or removing a dianionic phosphate group somewhere on a protein often changes the protein’s structural properties, its stability and dynamics. Moreover, the majority of signaling pathways involve an extensive set of protein-protein interactions, and phosphorylation can be used to regulate and modulate protein-protein binding. Losses of phosphorylation sites, as a result of disease mutations, might disrupt protein binding and deregulate signal transduction. In this paper we focus on the effects of phosphorylation on protein stability, dynamics and binding. We describe several physico-chemical mechanisms of protein regulation through phosphorylation and pay particular attention to phosphorylation in protein complexes and phosphorylation in the context of disorder-order and order-disorder transitions. Finally we assess the role of multiple phosphorylation sites in a protein molecule, their possible cooperativity and function.

  13. Transcriptional regulation by Polycomb group proteins

    DEFF Research Database (Denmark)

    Di Croce, Luciano; Helin, Kristian

    2013-01-01

    ) 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.......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...

  14. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

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

    Science.gov (United States)

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

    2008-05-13

    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 processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.

  16. 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

  17. Regulation of longevity by regulator of G-protein signaling protein, Loco.

    Science.gov (United States)

    Lin, Yuh-Ru; Kim, Keetae; Yang, Yanfei; Ivessa, Andreas; Sadoshima, Junichi; Park, Yongkyu

    2011-06-01

    Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

  18. Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation.

    Directory of Open Access Journals (Sweden)

    Benjamin M Laitman

    Full Text Available Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1 display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin Kpna4 is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. Kpna4 inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.

  19. The insulator protein CTCF regulates Drosophila steroidogenesis

    Directory of Open Access Journals (Sweden)

    Ujué Fresán

    2015-07-01

    Full Text Available The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis.

  20. Transcription regulation by the human Ccr4-Not proteins

    NARCIS (Netherlands)

    Zwartjes, Catharina Geertruida Maria

    2004-01-01

    Transcription by RNA polymerase II is a highly regulated process. Multiple protein complexes are involved in the regulation of mRNA synthesis. The Ccr4-Not complex regulates transcription at a global level and, most likely, requires other proteins to associate with promoters. The complex is

  1. A General Method for Regulating Protein Stability with Light

    OpenAIRE

    Bonger, Kimberly M; Rakhit, Rishi; Payumo, Alexander Y.; Chen, James K; Wandless, Thomas J.

    2013-01-01

    Posttranslational regulation of protein abundance in cells is a powerful tool for studying protein function. We here describe a novel genetically encoded protein domain that is degraded upon exposure to non-toxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

  2. 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.

  3. 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.

  4. 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

    proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  5. Regulation of Ebola virus VP40 matrix protein by SUMO

    National Research Council Canada - National Science Library

    Maite Baz-martínez; Ahmed El Motiam; Paula Ruibal; Gabriela N Condezo; Carlos F De La Cruz-herrera; Valerie Lang; Manuel Collado; Carmen San Martín; Manuel S Rodríguez; Cesar Muñoz-fontela; Carmen Rivas

    2016-01-01

    The matrix protein of Ebola virus (EBOV) VP40 regulates viral budding, nucleocapsid recruitment, virus structure and stability, viral genome replication and transcription, and has an intrinsic ability to form virus-like particles...

  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 func...

  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 func...

  8. 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.

  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...... to a common surface on PCNA; hence these interactions need to be tightly regulated and coordinated to ensure proper chromosome replication and integrity. Control of PCNA-protein interactions is multilayered and involves post-translational modifications, in particular ubiquitylation, accessory factors...... and regulated degradation of PCNA-associated proteins. This regulatory framework allows cells to maintain a fine-tuned balance between replication fidelity and processivity in response to DNA damage....

  10. Protein feature based identification of cell cycle regulated proteins in yeast

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Jensen, Lars Juhl

    2003-01-01

    DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features...... include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell...... cycle regulated proteins were identified, many of which have no known function....

  11. RNA-processing protein TDP-43 regulates FOXO-dependent protein quality control in stress response.

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2014-10-01

    Full Text Available Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43 is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases.

  12. 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.

  13. The Role of Bromodomain Proteins in Regulating Gene Expression

    Directory of Open Access Journals (Sweden)

    Michael F. Duffy

    2012-05-01

    Full Text Available Histone modifications are important in regulating gene expression in eukaryotes. Of the numerous histone modifications which have been identified, acetylation is one of the best characterised and is generally associated with active genes. Histone acetylation can directly affect chromatin structure by neutralising charges on the histone tail, and can also function as a binding site for proteins which can directly or indirectly regulate transcription. Bromodomains specifically bind to acetylated lysine residues on histone tails, and bromodomain proteins play an important role in anchoring the complexes of which they are a part to acetylated chromatin. Bromodomain proteins are involved in a diverse range of functions, such as acetylating histones, remodeling chromatin, and recruiting other factors necessary for transcription. These proteins thus play a critical role in the regulation of transcription.

  14. Effective identification of negative regulation patterns of protein kinases.

    Science.gov (United States)

    Chen, Qingfeng; Hu, Xiaoyan; Chen, Baoshan

    2013-06-01

    Recent studies point to the fact that protein kinases play an important role in the regulation of cellular pathways and show great potential in disease treatment. Thus, it is critical to discover characterized regulatory patterns of protein kinases in signaling pathway. There have been considerable efforts to explore the activities of protein kinases. However, the study of negative regulation patterns has been largely overlooked and undeveloped. This paper aims to identify inhibitory regulatory correlations of protein kinase according to negative association rule mining. Especially, mutual information is applied to sort out the items with strong dependency and the minimum support threshold is computed by support constraints to control rule generation. The obtained rules not only reveal the relationships between subunits of protein kinases and between subunits and stimuli, but also provide novel pharmacological insight into drug design for diseases.

  15. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    Science.gov (United States)

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. Copyright © 2016. Published by Elsevier Ltd.

  16. New Insights into the Protein Turnover Regulation in Ethylene Biosynthesis.

    Science.gov (United States)

    Yoon, Gyeong Mee

    2015-07-01

    Biosynthesis of the phytohormone ethylene is under tight regulation to satisfy the need for appropriate levels of ethylene in plants in response to exogenous and endogenous stimuli. The enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyzes the rate-limiting step of ethylene biosynthesis, plays a central role to regulate ethylene production through changes in ACS gene expression levels and the activity of the enzyme. Together with molecular genetic studies suggesting the roles of post-translational modification of the ACS, newly emerging evidence strongly suggests that the regulation of ACS protein stability is an alternative mechanism that controls ethylene production, in addition to the transcriptional regulation of ACS genes. In this review, recent new insight into the regulation of ACS protein turnover is highlighted, with a special focus on the roles of phosphorylation, ubiquitination, and novel components that regulate the turnover of ACS proteins. The prospect of cross-talk between ethylene biosynthesis and other signaling pathways to control turnover of the ACS protein is also considered.

  17. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    Science.gov (United States)

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    Science.gov (United States)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  19. Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination.

    Directory of Open Access Journals (Sweden)

    Santosh Kumar

    2011-01-01

    Full Text Available p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

  20. Developmental regulation of RNA processing by Rbfox proteins.

    Science.gov (United States)

    Conboy, John G

    2017-03-01

    The Rbfox genes encode an ancient family of sequence-specific RNA binding proteins (RBPs) that are critical developmental regulators in multiple tissues including skeletal muscle, cardiac muscle, and brain. The hallmark of Rbfox proteins is a single high-affinity RRM domain, highly conserved from insects to humans, that binds preferentially to UGCAUG motifs at diverse regulatory sites in pre-mRNA introns, mRNA 3'UTRs, and pre-miRNAs hairpin structures. Versatile regulatory circuits operate on Rbfox pre-mRNA and mRNA to ensure proper expression of Rbfox1 protein isoforms, which then act on the broader transcriptome to regulate alternative splicing networks, mRNA stability and translation, and microRNA processing. Complex Rbfox expression is encoded in large genes encompassing multiple promoters and alternative splicing options that govern spatiotemporal expression of structurally distinct and tissue-specific protein isoforms with different classes of RNA targets. Nuclear Rbfox1 is a candidate master regulator that binds intronic UGCAUG elements to impact splicing efficiency of target alternative exons, many in transcripts for other splicing regulators. Tissue-specificity of Rbfox-mediated alternative splicing is executed by combinatorial regulation through the integrated activity of Rbfox proteins and synergistic or antagonistic splicing factors. Studies in animal models show that Rbfox1-related genes are critical for diverse developmental processes including germ cell differentiation and memory in Drosophila, neuronal migration and function in mouse brain, myoblast fusion and skeletal muscle function, and normal heart function. Finally, genetic and biochemical evidence suggest that aberrations in Rbfox-regulated circuitry are risk factors for multiple human disorders, especially neurodevelopmental disorders including epilepsy and autism, and cardiac hypertrophy. WIREs RNA 2017, 8:e1398. doi: 10.1002/wrna.1398 For further resources related to this article, please

  1. Regulation of transferrin receptor 2 protein levels by transferrin.

    Science.gov (United States)

    Robb, Aeisha; Wessling-Resnick, Marianne

    2004-12-15

    Transferrin receptor 2 (TfR2) plays a critical role in iron homeostasis because patients carrying disabling mutations in the TFR2 gene suffer from hemochromatosis. In this study, iron-responsive regulation of TfR2 at the protein level was examined in vitro and in vivo. HepG2 cell TfR2 protein levels were up-regulated after exposure to holotransferrin (holoTf) in a time- and dose-responsive manner. ApoTf or high-iron treatment with non-Tf-bound iron failed to elicit similar effects, suggesting that TfR2 regulation reflects interactions of the iron-bound ligand. Hepatic TfR2 protein levels also reflected an adaptive response to changing iron status in vivo. Liver TfR2 protein levels were down- and up-regulated in rats fed an iron-deficient and a high-iron diet, respectively. TfR2 was also up-regulated in Hfe(-/-) mice, an animal model that displays liver iron loading. In contrast, TfR2 levels were reduced in hypotransferrinemic mice despite liver iron overload, supporting the idea that regulation of the receptor is dependent on Tf. This idea is confirmed by up-regulation of TfR2 in beta-thalassemic mice, which, like hypotransferrinemic mice, are anemic and incur iron loading, but have functional Tf. Based on these combined results, we hypothesize that TfR2 acts as a sensor of iron status such that receptor levels reflect Tf saturation.

  2. Regulation of Insulin Receptor Trafficking by Bardet Biedl Syndrome Proteins.

    Directory of Open Access Journals (Sweden)

    Rachel D Starks

    2015-06-01

    Full Text Available Insulin and its receptor are critical for the regulation of metabolic functions, but the mechanisms underlying insulin receptor (IR trafficking to the plasma membrane are not well understood. Here, we show that Bardet Biedl Syndrome (BBS proteins are necessary for IR localization to the cell surface. We demonstrate that the IR interacts physically with BBS proteins, and reducing the expression of BBS proteins perturbs IR expression in the cell surface. We show the consequence of disrupting BBS proteins for whole body insulin action and glucose metabolism using mice lacking different BBS genes. These findings demonstrate the importance of BBS proteins in underlying IR cell surface expression. Our data identify defects in trafficking and localization of the IR as a novel mechanism accounting for the insulin resistance commonly associated with human BBS. This is supported by the reduced surface expression of the IR in fibroblasts derived from patients bearing the M390R mutation in the BBS1 gene.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Regulation of lipid metabolism by angiopoietin-like proteins

    NARCIS (Netherlands)

    Dijk, Wieneke; Kersten, Sander

    2016-01-01

    PURPOSE OF REVIEW: The angiopoietin-like proteins (ANGPTLs) 3, 4 and 8 have emerged as key regulators of plasma lipid metabolism by serving as potent inhibitors of the enzyme lipoprotein lipase (LPL). In this review, we provide an integrated picture of the role of ANGPTL3, ANGPTL4 and ANGPTL8 in

  5. Regulation of protein activity with small-molecule-controlled inteins

    Science.gov (United States)

    Skretas, Georgios; Wood, David W.

    2005-01-01

    Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular “switches” for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology. PMID:15632292

  6. 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.

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

    Science.gov (United States)

    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.

  8. BCL-2 family proteins as regulators of mitochondria metabolism.

    Science.gov (United States)

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Sox proteins: regulators of cell fate specification and differentiation.

    Science.gov (United States)

    Kamachi, Yusuke; Kondoh, Hisato

    2013-10-01

    Sox transcription factors play widespread roles during development; however, their versatile funtions have a relatively simple basis: the binding of a Sox protein alone to DNA does not elicit transcriptional activation or repression, but requires binding of a partner transcription factor to an adjacent site on the DNA. Thus, the activity of a Sox protein is dependent upon the identity of its partner factor and the context of the DNA sequence to which it binds. In this Primer, we provide an mechanistic overview of how Sox family proteins function, as a paradigm for transcriptional regulation of development involving multi-transcription factor complexes, and we discuss how Sox factors can thus regulate diverse processes during development.

  10. Heat Shock Proteins in Tendinopathy: Novel Molecular Regulators

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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...... cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been......The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT...

  13. G protein-coupled receptors as regulators of energy homeostasis.

    Science.gov (United States)

    Tao, Ya-Xiong; Yuan, Zong-Hui; Xie, Jun

    2013-01-01

    G protein-coupled receptors (GPCRs) are versatile regulators of physiological processes. They are also important drug targets. Many of the molecules controlling energy homeostasis act through GPCRs. This article summarizes the regulators of energy homeostasis in the central nervous system: those secreted by the gastrointestinal peptides and those secreted by the endocrine pancreas. Some examples of orphan GPCRs are also given. The regulation of energy homeostasis is conserved in other mammals, including those species relevant in veterinary medicine, and fish. Finally, the genetics of human obesity is briefly summarized. Genetic susceptibility in the current obesogenic environment is likely causing the obesity pandemic. A better understanding of the regulation of energy homeostasis will lead to novel pharmacotherapy for obesity treatment. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. 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

  15. Regulation of dynamin family proteins by post-translational modifications.

    Science.gov (United States)

    Kar, Usha P; Dey, Himani; Rahaman, Abdur

    2017-06-01

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.

  16. Fluctuations within folded proteins: implications for thermodynamic and allosteric regulation.

    Science.gov (United States)

    DuBay, Kateri H; Bowman, Gregory R; Geissler, Phillip L

    2015-04-21

    Folded protein structures are both stable and dynamic. Historically, our clearest window into these structures came from X-ray crystallography, which generally provided a static image of each protein's singular "folded state", highlighting its stability. Deviations away from that crystallographic structure were difficult to quantify, and as a result, their potential functional consequences were often neglected. However, several dynamical and statistical studies now highlight the structural variability that is present within the protein's folded state. Here we review mounting evidence of the importance of these structural rearrangements; both experiment and computation indicate that folded proteins undergo substantial fluctuations that can greatly influence their function. Crucially, recent studies have shown that structural elements of proteins, especially their side-chain degrees of freedom, fluctuate in ways that generate significant conformational heterogeneity. The entropy associated with these motions contributes to the folded structure's thermodynamic stability. In addition, since these fluctuations can shift in response to perturbations such as ligand binding, they may play an important role in the protein's capacity to respond to environmental cues. In one compelling example, the entropy associated with side-chain fluctuations contributes significantly to regulating the binding of calmodulin to a set of peptide ligands. The neglect of fluctuations within proteins' native states was often justified by the dense packing within folded proteins, which has inspired comparisons with crystalline solids. Many liquids, however, can achieve similarly dense packing yet fluidity is maintained through correlated molecular motions. Indeed, the studies we discuss favor comparison of folded proteins not with solids but instead with dense liquids, where the internal side chain fluidity is facilitated by collective motions that are correlated over long distances. These

  17. The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.

    Science.gov (United States)

    Pelisch, Federico; Gerez, Juan; Druker, Jimena; Schor, Ignacio E; Muñoz, Manuel J; Risso, Guillermo; Petrillo, Ezequiel; Westman, Belinda J; Lamond, Angus I; Arzt, Eduardo; Srebrow, Anabella

    2010-09-14

    Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

  18. Transcriptional regulation of the uncoupling protein-1 gene.

    Science.gov (United States)

    Villarroya, Francesc; Peyrou, Marion; Giralt, Marta

    2017-03-01

    Regulated transcription of the uncoupling protein-1 (UCP1) gene, and subsequent UCP1 protein synthesis, is a hallmark of the acquisition of the differentiated, thermogenically competent status of brown and beige/brite adipocytes, as well as of the responsiveness of brown and beige/brite adipocytes to adaptive regulation of thermogenic activity. The 5' non-coding region of the UCP1 gene contains regulatory elements that confer tissue specificity, differentiation dependence, and neuro-hormonal regulation to UCP1 gene transcription. Two main regions-a distal enhancer and a proximal promoter region-mediate transcriptional regulation through interactions with a plethora of transcription factors, including nuclear hormone receptors and cAMP-responsive transcription factors. Co-regulators, such as PGC-1α, play a pivotal role in the concerted regulation of UCP1 gene transcription. Multiple interactions of transcription factors and co-regulators at the promoter region of the UCP1 gene result in local chromatin remodeling, leading to activation and increased accessibility of RNA polymerase II and subsequent gene transcription. Moreover, a commonly occurring A-to-G polymorphism in close proximity to the UCP1 gene enhancer influences the extent of UCP1 gene transcription. Notably, it has been reported that specific aspects of obesity and associated metabolic diseases are associated with human population variability at this site. On another front, the unique properties of the UCP1 promoter region have been exploited to develop brown adipose tissue-specific gene delivery tools for experimental purposes. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  19. Post-translational regulation and modifications of flavivirus structural proteins.

    Science.gov (United States)

    Roby, Justin A; Setoh, Yin Xiang; Hall, Roy A; Khromykh, Alexander A

    2015-07-01

    Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics.

  20. The role of Cockayne Syndrome Protein B in transcription regulation.

    Science.gov (United States)

    Jeong, Jieun

    2014-12-01

    We investigated the question if CSB (Cockayne Syndrome complementation B) protein actively regulates gene transcription and how mutations in CSB gene affect that regulatory role. Here we describe how we processed and interpreted ChIP-seq data (deposited in Gene Expression Omnibus with accession number GSE50171) obtained during an investigation of that question, and how this analysis assisted in the generation of hypothesis that were subsequently validated using other types of experiment.

  1. The role of Cockayne Syndrome Protein B in transcription regulation

    OpenAIRE

    Jeong, Jieun

    2014-01-01

    We investigated the question if CSB (Cockayne Syndrome complementation B) protein actively regulates gene transcription and how mutations in CSB gene affect that regulatory role. Here we describe how we processed and interpreted ChIP-seq data (deposited in Gene Expression Omnibus with accession number GSE50171) obtained during an investigation of that question, and how this analysis assisted in the generation of hypothesis that were subsequently validated using other types of experiment.

  2. Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst).

    Science.gov (United States)

    Karlgren, Anna; Gyllenstrand, Niclas; Källman, Thomas; Lagercrantz, Ulf

    2013-01-01

    From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.

  3. Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst.

    Directory of Open Access Journals (Sweden)

    Anna Karlgren

    Full Text Available From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana, a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1, GIGANTEA (GI, ZEITLUPE (ZTL and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1 that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1 in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partly conserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of the three-loop network typical of the circadian clock in angiosperms were present before the split of gymnosperms and angiosperms.

  4. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    Science.gov (United States)

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  5. Redox regulation by reversible protein S-thiolation in bacteria

    Directory of Open Access Journals (Sweden)

    Vu Van Loi

    2015-03-01

    Full Text Available Low molecular weight (LMW thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH while Actinomycetes produce the related redox buffer mycothiol (MSH. In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx. In addition, novel bacilliredoxins (Brx and mycoredoxins (Mrx1 were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria.

  6. Heat Shock Protein 90 regulates encystation in Entamoeba

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    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.

  7. Heat Shock Protein 90 regulates encystation in Entamoeba

    Science.gov (United States)

    Singh, Meetali; Sharma, Shalini; Bhattacharya, Alok; Tatu, Utpal

    2015-01-01

    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. PMID:26528271

  8. 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

    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...

  9. FEM1 proteins are ancient regulators of SLBP degradation.

    Science.gov (United States)

    Dankert, John F; Pagan, Julia K; Starostina, Natalia G; Kipreos, Edward T; Pagano, Michele

    2017-03-19

    FEM1A, FEM1B, and FEM1C are evolutionarily-conserved VHL-box proteins, the substrate recognition subunits of CUL2-RING E3 ubiquitin ligase complexes. Here, we report that FEM1 proteins are ancient regulators of Stem-Loop Binding Protein (SLBP), a conserved protein that interacts with the stem loop structure located in the 3' end of canonical histone mRNAs and functions in mRNA cleavage, translation and degradation. SLBP levels are highest during S-phase coinciding with histone synthesis. The ubiquitin ligase complex SCF(cyclin F) targets SLBP for degradation in G2 phase; however, the regulation of SLBP during other stages of the cell cycle is poorly understood. We provide evidence that FEM1A, FEM1B, and FEM1C interact with and mediate the degradation of SLBP. Cyclin F, FEM1A, FEM1B and FEM1C all interact with a region in SLBP's N-terminus using distinct degrons. An SLBP mutant that is unable to interact with all 4 ligases is expressed at higher levels than wild type SLBP and does not oscillate during the cell cycle. We demonstrate that orthologues of SLBP and FEM1 proteins interact in C. elegans and D. melanogaster, suggesting that the pathway is evolutionarily conserved. Furthermore, we show that FEM1 depletion in C. elegans results in the upregulation of SLBP ortholog CDL-1 in oocytes. Notably, cyclin F is absent in flies and worms, suggesting that FEM1 proteins play an important role in SLBP targeting in lower eukaryotes.

  10. Forkhead box protein A2 (FOXA2 protein stability and activity are regulated by sumoylation.

    Directory of Open Access Journals (Sweden)

    Narasimhaswamy S Belaguli

    Full Text Available The forkhead box protein A2 (FOXA2 is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1 and mapped the sumoylation site to the amino acid lysine 6 (K6. Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  11. 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.

  12. A dynamic response regulator protein modulates G-protein-dependent polarity in the bacterium Myxococcus xanthus.

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    Yong Zhang

    Full Text Available Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz. It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP. In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins.

  13. Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

    Science.gov (United States)

    Farhy Tselnicker, Isabella; Tsemakhovich, Vladimir; Rishal, Ida; Kahanovitch, Uri; Dessauer, Carmen W; Dascal, Nathan

    2014-04-01

    Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li(+) did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gβγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

  14. Regulation of protein synthesis during sea urchin early development

    Energy Technology Data Exchange (ETDEWEB)

    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. ({sup 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.

  15. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    Science.gov (United States)

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

  16. Matrix gla protein: An extracellular matrix protein regulates myostatin expression in the muscle developmental program.

    Science.gov (United States)

    Ahmad, Sarafraz; Jan, Arif Tasleem; Baig, Mohammad Hassan; Lee, Eun Ju; Choi, Inho

    2017-03-01

    Skeletal muscle development involves interactions between intracellular and extracellular factors that act in concert to regulate the myogenic process. Matrix gla protein (MGP), a well-known inhibitor of calcification in soft tissues, has been reported to be highly up-regulated during myogenesis. Our interest in the regulation of muscle satellite cells (MSCs) by extracellular matrix (ECM) led us to investigate the effects of MGP during the progression of myogenesis. Participation of MGP in the myogenic process was investigated in vitro using C2C12 cells, and knockdown of its gene was performed to determine its effects on the expression of myogenic regulatory factors (MRFs) and other ECM genes. In addition, interactions between MGP, Fibromodulin (FMOD), and Myostatin (MSTN) were investigated by conducting co-immunoprecipitation and in silico studies. Matrix gla protein knockdown (MGPkd) shows pronounced effects during myogenesis as evidenced by the down regulation of myogenic marker (MYOG and MYOD), and ECM (COL1α1 and FMOD) genes. Down-regulation of MSTN expression in MGPkd cells suggests its role in coordinating the regulation of MSTN expression. Having strong affinity for ACVRIIB receptor, in silico data confirms MGP interference in the interaction of MSTN with ACVRIIB. These findings show MGP inhibits MSTN functionally by disrupting its binding to receptor. The present study provides insights of an ECM protein that participates in the regulation of the myogenic program by inhibiting the activity of the myogenic negative regulator MSTN, which suggests that MGP might be used for designing novel inhibitors that can promote muscle regeneration or treat muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Studying allosteric regulation in metal sensor proteins using computational methods.

    Science.gov (United States)

    Chakravorty, Dhruva K; Merz, Kenneth M

    2014-01-01

    In this chapter, we describe advances made in understanding the mechanism of allosteric regulation of DNA operator binding in the ArsR/SmtB family of metal-sensing proteins using computational methods. The paradigm, zinc-sensing transcriptional repressor Staphylococcus aureus CzrA represents an excellent model system to understand how metal sensor proteins maintain cellular metal homeostasis. Here, we discuss studies that helped to characterize a metal ion-mediated hydrogen-bonding pathway (HBP) that plays a dominant role in the allosteric mechanism of DNA operator binding in these proteins. The chapter discusses computational methods used to provide a molecular basis for the large conformational motions and allosteric coupling free energy (~6kcal/mol) associated with Zn(II) binding in CzrA. We present an accurate and convenient means by which to include metal ions in the nuclear magnetic resonance (NMR) structure determination process using molecular dynamics (MD) constrained by NMR-derived data. The method provides a realistic and physically viable description of the metal-binding site(s) and has potentially broad applicability in the structure determination of metal ion-bound proteins, protein folding, and metal template protein-design studies. Finally, our simulations provide strong support for a proposed HBP that physically connects the metal-binding residue, His97, to the DNA-binding interface through the αR helix that is present only in the Zn(II)-bound state. We find the interprotomer hydrogen bond interaction to be significantly stronger (~8kcal/mol) at functional allosteric metal-binding sites compared to the apo proteins. This interaction works to overcome the considerable disorder at these hydrogen-bonding sites in apo protein and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. This interaction is found to be considerably weaker in nonresponsive metal-binding sites. These findings suggest a conserved functional

  18. Testosterone Regulates Tight Junction Proteins and Influences Prostatic Autoimmune Responses

    Science.gov (United States)

    Meng, Jing; Mostaghel, Elahe A.; Vakar-Lopez, Funda; Montgomery, Bruce; True, Larry; Nelson, Peter S.

    2015-01-01

    Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology. PMID:21761342

  19. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    Science.gov (United States)

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  20. Protein kinase D regulates cell death pathways in experimental pancreatitis

    Directory of Open Access Journals (Sweden)

    Jingzhen eYuan

    2012-03-01

    Full Text Available Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-kappaB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK in pancreatic acinar cells. Conversely, upregulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins (IAPs, caspases, receptor-interacting protein kinase 1 (RIP1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.

  1. 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

  2. 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-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator...... of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFβ is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear...

  3. Cathepsins B and L differentially regulate amyloid precursor protein processing.

    Science.gov (United States)

    Klein, Donna M; Felsenstein, Kevin M; Brenneman, Douglas E

    2009-03-01

    Previous studies have shown that cathepsins control amyloid beta (Abeta) levels in chromaffin cells via a regulated secretory pathway. In the present study, this concept was extended to investigations in primary hippocampal neurons to test whether Abeta release was coregulated by cathepsins and electrical activity, proposed components of a regulated secretory pathway. Inhibition of cathepsin B (catB) activity with CA074Me or attenuation of catB expression through small interfering RNA produced decreases in Abeta release, similar to levels produced with suppression of beta-site APP-cleaving enzyme 1 (BACE1) expression. To test whether the catB-dependent release of Abeta was linked to ongoing electrical activity, neurons were treated with tetrodotoxin (TTX) and CA074Me. These comparisons demonstrated no additivity between decreases in Abeta release produced by TTX and CA074Me. In contrast, pharmacological inhibition of cathepsin L (catL) selectively elevated Abeta42 levels but not Abeta40 or total Abeta. Mechanistic studies measuring C-terminal fragments of amyloid precursor protein (APP) suggested that catL elevated alpha-secretase activity, thereby suppressing Abeta42 levels. The mechanism of catB-mediated regulation of Abeta release remains unclear but may involve elevation of beta-secretase. In summary, these studies provide evidence for a significant alternative pathway for APP processing that involves catB and activity-dependent release of Abeta in a regulated secretory pathway for primary neurons.

  4. 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

  5. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    Arsenic is a metalloid that is toxic to living organisms. The use of arsenic-contaminated ground water for drinking and for irrigation in agriculture presents serious health problems for millions of people in many parts of the world. Arsenate (As(V)) and arsenite (As(III)), the two most widespread...... 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...... vacuoles. In this study using Arabidopsis, the role of TIP subfamily in arsenic transport was examined together with the role of N-terminus in regulation of AtNIP5;1, which has previously been shown to transport As(III) in a yeast expression system. The results showed that AtTIP4;1 functions...

  6. FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

    Science.gov (United States)

    Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

    2017-04-11

    The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice

    Directory of Open Access Journals (Sweden)

    Arisa Hirano

    2017-04-01

    Full Text Available The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD binding pocket of CRYPTOCHROME2 (CRY2, a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk, an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis. We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals.

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

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    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. Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Guanggan Hu

    2007-03-01

    Full Text Available A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP-dependent protein kinase A (PKA is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential

  10. 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.

  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. 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

  13. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    Science.gov (United States)

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  14. 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

  15. 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

  16. 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

    2017-10-16

    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.Laboratory Investigation advance online publication, 16 October 2017; doi:10.1038/labinvest.2017.103.

  17. The regulation of protein content and quality in national and international food standards.

    Science.gov (United States)

    Lewis, Janine L

    2012-08-01

    Food regulation aims to protect public health through a safe and nutritious food supply produced by a compliant food industry. Food standards of developed countries generally do not regulate protein content or protein quality because the risk of dietary protein inadequacy in their national populations is very low. Protein is nevertheless regulated for reasons of product quality or protein labelling or to minimise assessed health risks associated with consumption of certain animal- and vegetable-protein foods; analogue products that extend or simulate commonly available animal-protein foods; and special purpose foods such as infant formula and foods, supplementary and medical foods, and foods for weight loss. The extent and approach to protein regulation varies greatly among jurisdictions but where it occurs, it is applied through minimum and sometimes maximum limits on protein content or quality measures or both using an inter-related approach. Protein quality measures range from amino acid profiles and digestibility corrected scores to protein rating, a rat bioassay and reference proteins not further described. Regulatory methods for protein quality determination are referenced to the published scientific literature or developed nationally. Internationally, the Codex Alimentarius regulates the protein content and quality of some foods. The Codex approach varies according to the food but is similar to the approaches used in national and regional food regulation. This paper provides a comparison of the regulation of protein in foods using examples from the food regulations of Australia New Zealand, Canada, the European Union, the United States of America and the Codex Alimentarius.

  18. Protein kinase C signaling and cell cycle regulation

    Science.gov (United States)

    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 30 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 T cells. PMID

  19. 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.

  20. Myeloid-related protein-14 regulates deep vein thrombosis

    Science.gov (United States)

    Wang, Yunmei; Gao, Huiyun; Kessinger, Chase W.; Schmaier, Alvin; Jaffer, Farouc A.; Simon, Daniel I.

    2017-01-01

    Using transcriptional profiling of platelets from patients presenting with acute myocardial infarction, we identified myeloid-related protein-14 (MRP-14, also known as S100A9) as an acute myocardial infarction gene and reported that platelet MRP-14 binding to platelet CD36 regulates arterial thrombosis. However, whether MRP-14 plays a role in venous thrombosis is unknown. We subjected WT and Mrp-14–deficient (Mrp-14-/-) mice to experimental models of deep vein thrombosis (DVT) by stasis ligation or partial flow restriction (stenosis) of the inferior vena cava. Thrombus weight in response to stasis ligation or stenosis was reduced significantly in Mrp-14-/- mice compared with WT mice. The adoptive transfer of WT neutrophils or platelets, or the infusion of recombinant MRP-8/14, into Mrp-14-/- mice rescued the venous thrombosis defect in Mrp-14-/- mice, indicating that neutrophil- and platelet-derived MRP-14 directly regulate venous thrombogenesis. Stimulation of neutrophils with MRP-14 induced neutrophil extracellular trap (NET) formation, and NETs were reduced in venous thrombi harvested from Mrp-14-/- mice and in Mrp-14-/- neutrophils stimulated with ionomycin. Given prior evidence that MRP-14 also regulates arterial thrombosis, but not hemostasis (i.e., reduced bleeding risk), MRP-14 appears to be a particularly attractive molecular target for treating thrombotic cardiovascular diseases, including myocardial infarction, stroke, and venous thromboembolism. PMID:28570273

  1. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  2. Leptin and Fasting Regulate Rat Gastric Glucose-Regulated Protein 58

    Directory of Open Access Journals (Sweden)

    Susana B. Bravo

    2011-01-01

    Full Text Available The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58 is a molecular chaperone member of the endoplasmic reticulum (ER stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency. We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.

  3. Munc13 proteins control regulated exocytosis in mast cells.

    Science.gov (United States)

    Rodarte, Elsa M; Ramos, Marco A; Davalos, Alfredo J; Moreira, Daniel C; Moreno, David S; Cardenas, Eduardo I; Rodarte, Alejandro I; Petrova, Youlia; Molina, Sofia; Rendon, Luis E; Sanchez, Elizabeth; Breaux, Keegan; Tortoriello, Alejandro; Manllo, John; Gonzalez, Erika A; Tuvim, Michael J; Dickey, Burton F; Burns, Alan R; Heidelberger, Ruth; Adachi, Roberto

    2017-11-15

    Mast cells (MCs) are involved in host defenses against pathogen and inflammation. Stimulated MCs release substances stored in their granules via regulated exocytosis. In other cell types, Munc13 (mammalian homolog of C. elegans uncoordinated gene 13) proteins play essential roles in regulated exocytosis. Here, we found that MCs express Munc13-2 and -4, and studied their roles using global and conditional knockout (KO) mice. In a model of systemic anaphylaxis, we found no difference between WT and Munc13-2 KO mice, but global and MC-specific Munc13-4 KO mice developed less hypothermia. This protection correlated with lower plasma histamine levels and with histological evidence of defective MC degranulation, and not with changes in MC development, distribution, numbers or morphology. In vitro assays revealed that the defective response in Munc13-4-deficient MCs was limited to regulated exocytosis, leaving other MC secretory effector responses intact. Single cell capacitance measurements in MCs from mouse mutants differing in Munc13-4 expression levels in their MCs revealed that as levels of Munc13-4 decrease, the rate of exocytosis declines first, and then the total amount of exocytosis decreases. A requirement for Munc13-2 in MC exocytosis was revealed only in the absence of Munc13-4. Electrophysiology and EM studies uncovered that the number of multigranular compound events (i.e., granule-to-granule homotypic fusion) was severely reduced in the absence of Munc13-4. We conclude that while Munc13-2 plays a minor role, Munc13-4 is essential for regulated exocytosis in MCs, and that this MC effector response is required for a full anaphylactic response. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  4. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

    Directory of Open Access Journals (Sweden)

    Nirmalya Chatterjee

    2016-05-01

    Full Text Available Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.

  5. 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.

  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. 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.

  8. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    Science.gov (United States)

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development. 2005 S. Karger AG, Basel

  9. 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 th...... 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....... In this study, we investigated the function of another member of this family, FSP27 (Cidec), in apoptosis and adipocyte metabolism. Although overexpression of FSP27 is sufficient to increase apoptosis of 293T and 3T3-L1 cells, more physiological levels of expression stimulate spontaneous lipid accumulation...... 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...

  10. 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.

  11. 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.

  12. RAF protein-serine/threonine kinases: Structure and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Roskoski, Robert, E-mail: rrj@brimr.org [Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116, Box 19, Horse Shoe, NC 28742 (United States)

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  13. Mammalian Hippo signalling: a kinase network regulated by protein-protein interactions

    Science.gov (United States)

    Hergovich, Alexander

    2012-01-01

    The Hippo signal transduction cascade controls cell growth, proliferation, and death, all of which are frequently deregulated in tumour cells. Since initial studies in Drosophila melanogaster were instrumental in defining Hippo signalling, the machinery was named after the central Ste20-like kinase Hippo. Moreover, given that loss of Hippo signalling components Hippo, Warts, and Mats resulted in uncontrolled tissue overgrowth, Hippo signalling was defined as a tumour suppressor cascade. Significantly, all core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in flies. Furthermore, studies in flies and mammalian cell systems showed that Hippo signalling represents a kinase cascade that is tightly regulated by protein-protein interactions (PPIs). Several Hippo signalling molecules contain SARAH domains that mediate specific PPIs, thereby influencing the activities of MST1/2 kinases, the human Hippo orthologues. Moreover, WW domains are present in several Hippo factors, and these domains also serve as interaction surfaces for regulatory PPIs in Hippo signalling. Finally, the kinase activities of LATS1/2, the human counterparts of Warts, are controlled by binding to hMOB1, the human Mats. Therefore, Hippo signalling is regulated by PPIs on several levels. Here we review our current understanding of how these regulatory PPIs are regulated and contribute to the functionality of Hippo signalling. PMID:22260677

  14. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    Science.gov (United States)

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  15. Proteins in growth regulation during early development. Progress report, 1976--1977

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N.W.

    1977-08-01

    Studies were conducted to determine mechanisms regulating the synthesis of serum proteins by the yolk-sac and to determine if serum proteins are developmental signals. Synthesis of serum proteins in the yolk-sac and liver was determined at various stages of development and effects of teratogenic agents and genetic mutations on protein synthesis were studied. The regulation of serum protein synthesis was studied in cell cultures and in cell-free protein synthesizing systems derived from the yolk-sac. Studies were also conducted on effects of serum proteins on rat embryos, chick embryos without yolk-sacs, and isolated brains. (HLW)

  16. Diferric transferrin regulates transferrin receptor 2 protein stability.

    Science.gov (United States)

    Johnson, Martha B; Enns, Caroline A

    2004-12-15

    Transferrin receptor 2 (TfR2) is a type 2 transmembrane protein expressed in hepatocytes that binds iron-bound transferrin (Tf). Mutations in TfR2 cause one form of hereditary hemochromatosis, a disease in which excessive absorption of dietary iron can lead to liver cirrhosis, diabetes, arthritis, and heart failure. The function of TfR2 in iron homeostasis is unknown. We have studied the regulation of TfR2 in HepG2 cells. Western blot analysis shows that TfR2 increases in a time- and dose-dependent manner after diferric Tf is added to the culture medium. In cells exposed to diferric Tf, the amount of TfR2 returns to control levels within 8 hours after the removal of diferric Tf from the medium. However, TfR2 does not increase when non-Tf-bound iron (FeNTA) or apo Tf is added to the medium. The response to diferric Tf appears to be hepatocyte specific. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis shows that TfR2 mRNA levels do not change in cells exposed to diferric Tf. Rather, the increase in TfR2 is attributed to an increase in the half-life of TfR2 protein in cells exposed to diferric Tf. Our results support a role for TfR2 in monitoring iron levels by sensing changes in the concentration of diferric Tf.

  17. V-1 regulates capping protein activity in vivo.

    Science.gov (United States)

    Jung, Goeh; Alexander, Christopher J; Wu, Xufeng S; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A

    2016-10-25

    Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

  18. 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.

  19. 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.

  20. 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 ...

  1. MicroRNA-Regulated Protein-Protein Interaction Networks and Their Functions in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Yen-Jen Oyang

    2013-05-01

    Full Text Available MicroRNAs, which are small endogenous RNA regulators, have been associated with various types of cancer. Breast cancer is a major health threat for women worldwide. Many miRNAs were reported to be associated with the progression and carcinogenesis of breast cancer. In this study, we aimed to discover novel breast cancer-related miRNAs and to elucidate their functions. First, we identified confident miRNA-target pairs by combining data from miRNA target prediction databases and expression profiles of miRNA and mRNA. Then, miRNA-regulated protein interaction networks (PINs were constructed with confident pairs and known interaction data in the human protein reference database (HPRD. Finally, the functions of miRNA-regulated PINs were elucidated by functional enrichment analysis. From the results, we identified some previously reported breast cancer-related miRNAs and functions of the PINs, e.g., miR-125b, miR-125a, miR-21, and miR-497. Some novel miRNAs without known association to breast cancer were also found, and the putative functions of their PINs were also elucidated. These include miR-139 and miR-383. Furthermore, we validated our results by receiver operating characteristic (ROC curve analysis using our miRNA expression profile data, gene expression-based outcome for breast cancer online (GOBO survival analysis, and a literature search. Our results may provide new insights for research in breast cancer-associated miRNAs.

  2. The SRC-associated protein CUB Domain-Containing Protein-1 regulates adhesion and motility.

    Science.gov (United States)

    Benes, C H; Poulogiannis, G; Cantley, L C; Soltoff, S P

    2012-02-02

    Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.

  3. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  4. Differential cellular protein expression in continuous porcine alveolar macrophages regulated by the porcine reproductive and respiratory syndrome virus nucleocapsid protein.

    Science.gov (United States)

    Sagong, Mingeun; Lee, Changhee

    2010-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a leading cause of significant economic losses in the pig industry worldwide. PRRSV infects preferentially porcine alveolar macrophages (PAMs) and subsequently utilizes the host cell biosynthetic machinery for its own replication. To date, a number of studies have been conducted to investigate compensatory changes of cellular gene expression of PAMs upon PRRSV infection. However, very little information exists about differential cellular protein expression of the natural target cells regulated by each viral protein. This study was therefore designed to examine the dynamics of host protein expression of continuous PAM cells by the PRRSV nucleocapsid (N) protein that is the most abundant and multifunctional viral component. We first established sublines of PAM cells to stably express the PRRSV N protein and assessed alterations in cellular protein productions of N-expressing PAM (PAM-pCD163-N) cells at different time courses by the use of proteomic analysis. A total of 23 protein spots were initially found to be differentially expressed in PAM-pCD163-N cells compared with normal PAM cells by high-resolution two-dimensional gel electrophoresis (2DE). Of these spots, 15 protein spots with statistically significant alteration, including 4 up-regulated and 11 down-regulated protein spots, were picked out for subsequent protein identification by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). The altered cellular proteins identified in this study were classified into the functions involved in a variety of cellular processes such as cell division, metabolism, inflammation response, stress response, ubiquitin-proteasome pathway, protein folding and synthesis, and transportation. Notably, heat shock 27kDa protein (HSP27) was found to be up-regulated in PAM-pCD163-N cells. The proteomics data will provide insights into the specific

  5. Regulation of orange carotenoid protein activity in cyanobacterial photoprotection

    NARCIS (Netherlands)

    Thurotte, A.; Lopez Igual, R.; Wilson, A.; Comolet, L.; Bourcier de Carbon, C.; Xiao, F.; Kirilovsky, D.

    2015-01-01

    Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism.

  6. Regulation of dynamin family proteins by post-translational ...

    Indian Academy of Sciences (India)

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agentsinvolved in several biological processes such as endocytosis, maintenance of organelle morphology and viralresistance. These large GTPases couple GTP hydrolysis with membrane alterations such as ...

  7. Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation*

    Science.gov (United States)

    Jung, Min-Su; Park, Jung-Hwa; Ryu, Young Shin; Choi, Sun-Hee; Yoon, Song-Hee; Kwen, Mi-Yang; Oh, Ji Youn; Song, Woo-Joo; Chung, Sul-Hee

    2011-01-01

    Two genes on chromosome 21, namely dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) and regulator of calcineurin 1 (RCAN1), have been implicated in some of the phenotypic characteristics of Down syndrome, including the early onset of Alzheimer disease. Although a link between Dyrk1A and RCAN1 and the nuclear factor of activated T cells (NFAT) pathway has been reported, it remains unclear whether Dyrk1A directly interacts with RCAN1. In the present study, Dyrk1A is shown to directly interact with and phosphorylate RCAN1 at Ser112 and Thr192 residues. Dyrk1A-mediated phosphorylation of RCAN1 at Ser112 primes the protein for the GSK3β-mediated phosphorylation of Ser108. Phosphorylation of RCAN1 at Thr192 by Dyrk1A enhances the ability of RCAN1 to inhibit the phosphatase activity of calcineurin (Caln), leading to reduced NFAT transcriptional activity and enhanced Tau phosphorylation. These effects are mediated by the enhanced binding of RCAN1 to Caln and its extended half-life caused by Dyrk1A-mediated phosphorylation. Furthermore, an increased expression of phospho-Thr192-RCAN1 was observed in the brains of transgenic mice overexpressing the Dyrk1A protein. These results suggest a direct link between Dyrk1A and RCAN1 in the Caln-NFAT signaling and Tau hyperphosphorylation pathways, supporting the notion that the synergistic interaction between the chromosome 21 genes RCAN1 and Dyrk1A is associated with a variety of pathological features associated with DS. PMID:21965663

  8. Regulation of RCAN1 protein activity by Dyrk1A protein-mediated phosphorylation.

    Science.gov (United States)

    Jung, Min-Su; Park, Jung-Hwa; Ryu, Young Shin; Choi, Sun-Hee; Yoon, Song-Hee; Kwen, Mi-Yang; Oh, Ji Youn; Song, Woo-Joo; Chung, Sul-Hee

    2011-11-18

    Two genes on chromosome 21, namely dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) and regulator of calcineurin 1 (RCAN1), have been implicated in some of the phenotypic characteristics of Down syndrome, including the early onset of Alzheimer disease. Although a link between Dyrk1A and RCAN1 and the nuclear factor of activated T cells (NFAT) pathway has been reported, it remains unclear whether Dyrk1A directly interacts with RCAN1. In the present study, Dyrk1A is shown to directly interact with and phosphorylate RCAN1 at Ser(112) and Thr(192) residues. Dyrk1A-mediated phosphorylation of RCAN1 at Ser(112) primes the protein for the GSK3β-mediated phosphorylation of Ser(108). Phosphorylation of RCAN1 at Thr(192) by Dyrk1A enhances the ability of RCAN1 to inhibit the phosphatase activity of calcineurin (Caln), leading to reduced NFAT transcriptional activity and enhanced Tau phosphorylation. These effects are mediated by the enhanced binding of RCAN1 to Caln and its extended half-life caused by Dyrk1A-mediated phosphorylation. Furthermore, an increased expression of phospho-Thr(192)-RCAN1 was observed in the brains of transgenic mice overexpressing the Dyrk1A protein. These results suggest a direct link between Dyrk1A and RCAN1 in the Caln-NFAT signaling and Tau hyperphosphorylation pathways, supporting the notion that the synergistic interaction between the chromosome 21 genes RCAN1 and Dyrk1A is associated with a variety of pathological features associated with DS.

  9. 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.

  10. Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control

    OpenAIRE

    Han, Sumin; Lone, Museer A.; Schneiter, Roger; CHANG, Amy

    2010-01-01

    Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regul...

  11. A DEAD Box RNA Helicase Is Critical for Pre-mRNA Splicing, Cold-Responsive Gene Regulation, and Cold Tolerance in Arabidopsis[C][W

    Science.gov (United States)

    Guan, Qingmei; Wu, Jianmin; Zhang, Yanyan; Jiang, Changhua; Liu, Renyi; Chai, Chenglin; Zhu, Jianhua

    2013-01-01

    Cold stress resulting from chilling and freezing temperatures substantially reduces crop production worldwide. To identify genes critical for cold tolerance in plants, we screened Arabidopsis thaliana mutants for deregulated expression of a firefly luciferase reporter gene under the control of the C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC). A regulator of CBF gene expression1 (rcf1-1) mutant that is hypersensitive to cold stress was chosen for in-depth characterization. RCF1 encodes a cold-inducible DEAD (Asp-Glu-Ala-Asp) box RNA helicase. Unlike a previously reported DEAD box RNA helicase (LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES4 [LOS4]) that regulates mRNA export, RCF1 does not play a role in mRNA export. Instead, RCF1 functions to maintain proper splicing of pre-mRNAs; many cold-responsive genes are mis-spliced in rcf1-1 mutant plants under cold stress. Functional characterization of four genes (PSEUDO-RESPONSE REGULATOR5 [PRR5], SHAGGY-LIKE SERINE/THREONINE KINASE12 [SK12], MYB FAMILY TRANSCRIPTION FACTOR CIRCADIAN1 [CIR1], and SPFH/PHB DOMAIN-CONTAINING MEMBRANE-ASSOCIATED PROTEIN [SPFH]) that are mis-spliced in rcf1-1 revealed that these genes are cold-inducible positive (CIR1 and SPFH) and negative (PRR5 and SK12) regulators of cold-responsive genes and cold tolerance. Together, our results suggest that the cold-inducible RNA helicase RCF1 is essential for pre-mRNA splicing and is important for cold-responsive gene regulation and cold tolerance in plants. PMID:23371945

  12. 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.

  13. Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion.

    Science.gov (United States)

    Sallee, Jennifer L; Wittchen, Erika S; Burridge, Keith

    2006-06-16

    Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.

  14. 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.

    2015-01-01

    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,

  15. FAD regulates CRYPTOCHROME protein stability and circadian clock in mice

    OpenAIRE

    Arisa Hirano; Daniel Braas; Ying-Hui Fu; Ptáček, Louis J.

    2017-01-01

    The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin ki...

  16. Rationally designed peptide regulators of protein kinase C

    OpenAIRE

    Churchill, Eric N.; Qvit, Nir; Mochly-Rosen, Daria

    2008-01-01

    Protein-protein interactions sequester enzymes close to their substrates. Protein kinase C (PKC) is one example of a ubiquitous signaling molecule with effects that are dependent upon localization. Short peptides derived from interaction sites between each PKC isozyme and its receptor for activated C kinase act as highly specific inhibitors and have become available as selective drugs in basic research and animal models of human diseases, such as myocardial infarction and hyperglycemia. Where...

  17. Gene regulation in response to protein disulphide isomerase deficiency

    DEFF Research Database (Denmark)

    Nørgaard, Per; Tachibana, Christine; Bruun, Anette W

    2003-01-01

    We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue...... element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes....

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

    Indian Academy of Sciences (India)

    Modification of proteins by phosphorylation is the major general mechanism by which many cellular functions in eukaryotic cells such as cell division, malignant transfor- mation, differentiation, signal transduction etc. are controll- ed by external physiological stimuli. At the molecular level protein ...

  19. 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

    Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical entity...... 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...

  20. 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.

  1. 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.

  2. Composite functional module inference: detecting cooperation between transcriptional regulation and protein interaction by mantel test

    Directory of Open Access Journals (Sweden)

    Su Fei

    2010-06-01

    Full Text Available Abstract Background Functional modules are basic units of cell function, and exploring them is important for understanding the organization, regulation and execution of cell processes. Functional modules in single biological networks (e.g., the protein-protein interaction network, have been the focus of recent studies. Functional modules in the integrated network are composite functional modules, which imply the complex relationships involving multiple biological interaction types, and detect them will help us understand the complexity of cell processes. Results We aimed to detect composite functional modules containing co-transcriptional regulation interaction, and protein-protein interaction, in our pre-constructed integrated network of Saccharomyces cerevisiae. We computationally extracted 15 composite functional modules, and found structural consistency between co-transcriptional regulation interaction sub-network and protein-protein interaction sub-network that was well correlated with their functional hierarchy. This type of composite functional modules was compact in structure, and was found to participate in essential cell processes such as oxidative phosphorylation and RNA splicing. Conclusions The structure of composite functional modules containing co-transcriptional regulation interaction, and protein-protein interaction reflected the cooperation of transcriptional regulation and protein function implementation, and was indicative of their important roles in essential cell functions. In addition, their structural and functional characteristics were closely related, and suggesting the complexity of the cell regulatory system.

  3. IQGAP scaffold proteins are the multifunctional regulators of cellular signaling and malignant transformation

    Directory of Open Access Journals (Sweden)

    P. A. Skovorodnikova

    2017-01-01

    Full Text Available Scaffold proteins coordinate the assembling of multicomponent protein complexes and participate in transduction of cellular signals via multiple signaling pathways therefore acting as important regulators of cell properties. IQ Motif Containing GTPase Activating Proteins (IQGAPs are promising targets for studying the role of scaffold proteins in intracellular signaling regulation and development of cancer and other diseases. IQGAP family includes 3 proteins (IQGAP1, IQGAP2 and IQGAP3 that differ considerably by their expression patterns and functions. Distinct genomic aberrations and expression changes in various tumors were reported for all three IQGAP family members. The present paper thoroughly reviews the structure of IQGAP proteins, their involvement in regulation of cell characteristics and interactions with components of intracellular signaling pathways. Special attention is given to the up-to-date data on deregulation of IQGAP genes functions in different tumor types, analysis of their possible role in tumor progression and their associations with clinicopathological tumor characteristics.

  4. Structural Elements Regulating AAA+ Protein Quality Control Machines

    Directory of Open Access Journals (Sweden)

    Chiung-Wen Chang

    2017-05-01

    Full Text Available 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.

  5. The Zika virus envelope protein glycan loop regulates virion antigenicity.

    Science.gov (United States)

    Goo, Leslie; DeMaso, Christina R; Pelc, Rebecca S; Ledgerwood, Julie E; Graham, Barney S; Kuhn, Richard J; Pierson, Theodore C

    2018-01-02

    Because antibodies are an important component of flavivirus immunity, understanding the antigenic structure of flaviviruses is critical. Compared to dengue virus (DENV), the loop containing the single N-linked glycosylation site on Zika virus (ZIKV) envelope (E) proteins extends further towards the DII fusion loop (DII-FL) on neighboring E proteins within E dimers on mature viruses. Although ZIKV is poorly neutralized by DII-FL antibodies, we demonstrated significantly increased neutralization sensitivity of ZIKV particles incorporating the DENV glycan loop. Increased neutralization sensitivity was independent of E protein glycosylation: ZIKV lacking E protein glycans remained poorly neutralized, whereas ZIKV loop chimeras with or without an E protein glycan were potently neutralized. ZIKV particles lacking the E protein glycan were capable of infecting Raji cells expressing the lectin DC-SIGNR, suggesting the prM glycan of partially mature particles can facilitate entry. Our study provides insight into the determinants of ZIKV E protein function and antigenicity. Copyright © 2017. Published by Elsevier Inc.

  6. mTORC1 Dependent Regulation of REDD1 Protein Stability

    OpenAIRE

    Chia Yee Tan; Thilo Hagen

    2013-01-01

    REDD1 is known to be transcriptionally upregulated in hypoxia. During hypoxic stress, REDD1 plays an important role as a mediator of mTORC1 inhibition. REDD1 is also subject to highly dynamic transcriptional regulation in response to a variety of other stress signals. In addition, the REDD1 protein is highly unstable. However, it is currently not well understood how REDD1 protein stability is regulated. In this study, we discovered that mTORC1 regulates REDD1 protein stability in a 26S protea...

  7. Inactivation, stabilization and redox regulation of iron-containing proteins

    NARCIS (Netherlands)

    Spee, J.H.

    1997-01-01

    Summary

    Microperoxidases: kinetics and stability.

    Microperoxidases are small enzymes prepared by proteolytic digestion of cytochromes c. The proteolytic removal of most of the protein environment allows these enzymes to use a

  8. Regulation of autophagy by protein post-translational modification

    National Research Council Canada - National Science Library

    Wani, Willayat Yousuf; Boyer-Guittaut, Michaël; Dodson, Matthew; Chatham, John; Darley-Usmar, Victor; Zhang, Jianhua

    2015-01-01

    ... status, and has an important role in quality control of macromolecules and organelles. As with other major cellular pathways, autophagy proteins are subjected to regulatory post-translational modification...

  9. Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

    Science.gov (United States)

    Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2017-02-01

    Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Mimitin - a novel cytokine-regulated mitochondrial protein.

    Science.gov (United States)

    Wegrzyn, Paulina; Yarwood, Stephen J; Fiegler, Nathalie; Bzowska, Monika; Koj, Aleksander; Mizgalska, Danuta; Malicki, Stanisław; Pajak, Magdalena; Kasza, Aneta; Kachamakova-Trojanowska, Neli; Bereta, Joanna; Jura, Jacek; Jura, Jolanta

    2009-03-31

    The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin - a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18-24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of apoptotic caspases 3

  11. Mimitin – a novel cytokine-regulated mitochondrial protein

    Directory of Open Access Journals (Sweden)

    Kachamakova-Trojanowska Neli

    2009-03-01

    Full Text Available Abstract Background The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin – a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. Results By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18–24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. Conclusion Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S, and some changes of mimitin gene

  12. Mimitin – a novel cytokine-regulated mitochondrial protein

    Science.gov (United States)

    Wegrzyn, Paulina; Yarwood, Stephen J; Fiegler, Nathalie; Bzowska, Monika; Koj, Aleksander; Mizgalska, Danuta; Malicki, Stanisław; Pajak, Magdalena; Kasza, Aneta; Kachamakova-Trojanowska, Neli; Bereta, Joanna; Jura, Jacek; Jura, Jolanta

    2009-01-01

    Background The product of a novel cytokine-responsive gene discovered by differential display analysis in our earlier studies on HepG2 cells was identified as mimitin – a small mitochondrial protein. Since proinflammatory cytokines are known to affect components of the respiratory chain in mitochondria, and mimitin was reported as a possible chaperone for assembly of mitochondrial complex I, we looked for the effects of modulation of mimitin expression and for mimitin-binding partners. Results By blocking mimitin expression in HepG2 cells by siRNA we found that mimitin has no direct influence on caspase 3/7 activities implicated in apoptosis. However, when apoptosis was induced by TNF and cycloheximide, and mimitin expression blocked, the activities of these caspases were significantly increased. This was accompanied by a slight decrease in proliferation of HepG2 cells. Our observations suggest that mimitin may be involved in the control of apoptosis indirectly, through another protein, or proteins. Using the yeast two-hybrid system and coimmunoprecipitation we found MAP1S among proteins interacting with mimitin. MAP1S is a recently identified member of the microtubule-associated protein family and has been shown to interact with NADH dehydrogenase I and cytochrome oxidase I. Moreover, it was implicated in the process of mitochondrial aggregation and nuclear genome destruction. The expression of mimitin is stimulated more than 1.6-fold by IL-1 and by IL-6, with the maximum level of mimitin observed after 18–24 h exposure to these cytokines. We also found that the cytokine-induced signal leading to stimulation of mimitin synthesis utilizes the MAP kinase pathway. Conclusion Mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. Mimitin interacts with a microtubular protein (MAP1S), and some changes of mimitin gene expression modulate activity of

  13. Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals

    National Research Council Canada - National Science Library

    Mitsunori Miyazaki; Karyn A. Esser

    2009-01-01

    .... In this review, we discuss the animal and cell culture models used and the signaling mechanisms identified in understanding regulation of protein synthesis in response to mechanical loading/resistance exercise...

  14. 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.

  15. 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.

  16. 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 this

  17. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins

  18. Regulation of flowering time by FVE, a retinoblastoma-associated protein.

    Science.gov (United States)

    Ausín, Israel; Alonso-Blanco, Carlos; Jarillo, José A; Ruiz-García, Leonor; Martínez-Zapater, José M

    2004-02-01

    The initiation of flowering in plants is controlled by environmental and endogenous signals. Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering.

  19. Considering Protonation as a Post-translational Modification Regulating Protein Structure and Function

    Science.gov (United States)

    Schönichen, André; Webb, Bradley A.; Jacobson, Matthew P.; Barber, Diane L.

    2014-01-01

    Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes. PMID:23451893

  20. 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.

  1. Proteins regulating cyclin dependent kinases Cdk4 and Cdk5

    NARCIS (Netherlands)

    Moorthamer, M.J.M.W.

    1999-01-01

    The exact passage through the eukaryotic cell cycle is regulated by the progressive activation and inactivation of a family Cdk-s. Cancer cells evolve from normal cells when some essential processes in a dividing cell malfunction. This causes inappropriate replication, segregation and

  2. Protein Kinase Pathways That Regulate Neuronal Survival and Death

    Science.gov (United States)

    2004-08-01

    intracellular T. gondii replication. International Congress on Toxoplasmosis . Freising, Germany, 2001. 13. Linseman, DA, T Laessig, MK Meintzer, M...University of Colo- role in the regulation of metabolic pathways as well as preven- rado Cancer Center core facility. tion of cell death by insulin and

  3. A Mur Regulator Protein in the Extremophilic Bacterium Deinococcus radiodurans

    Science.gov (United States)

    Wang, Yunfei; Yan, Guoquan; Zhang, Qikun; Wang, Liangyan; Tian, Bing; Chen, Huan; Hua, Yuejin

    2014-01-01

    Ferric uptake regulator (Fur) is a transcriptional regulator that controls the expression of genes involved in the uptake of iron and manganese, as well as vital nutrients, and is essential for intracellular redox cycling. We identified a unique Fur homolog (DR0865) from Deinococcus radiodurans, which is known for its extreme resistance to radiation and oxidants. A dr0865 mutant (Mt-0865) showed a higher sensitivity to manganese stress, hydrogen peroxide, gamma irradiation and ultraviolet (UV) irradiation than the wild-type R1 strain. Cellular manganese (Mn) ion (Mn2+) analysis showed that Mn2+, copper (Cu2+), and ferric (Fe3+) ions accumulated significantly in the mutant, which suggests that the dr0865 gene is not only involved in the regulation of Mn2+ homeostasis, but also affects the uptake of other ions. In addition, transcriptome profiles under MnCl2 stress showed that the expression of many genes involved in Mn metabolism was significantly different in the wild-type R1 and DR0865 mutant (Mt-0865). Furthermore, we found that the dr0865 gene serves as a positive regulator of the manganese efflux pump gene mntE (dr1236), and as a negative regulator of Mn ABC transporter genes, such as dr2283, dr2284 and dr2523. Therefore, it plays an important role in maintaining the homoeostasis of intracellular Mn (II), and also other Mn2+, zinc (Zn2+) and Cu2+ ions. Based on its role in manganese homeostasis, DR0865 likely belongs to the Mur sub-family of Fur homolog. PMID:25243898

  4. A Mur regulator protein in the extremophilic bacterium Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Amir Miraj Ul Hussain Shah

    Full Text Available Ferric uptake regulator (Fur is a transcriptional regulator that controls the expression of genes involved in the uptake of iron and manganese, as well as vital nutrients, and is essential for intracellular redox cycling. We identified a unique Fur homolog (DR0865 from Deinococcus radiodurans, which is known for its extreme resistance to radiation and oxidants. A dr0865 mutant (Mt-0865 showed a higher sensitivity to manganese stress, hydrogen peroxide, gamma irradiation and ultraviolet (UV irradiation than the wild-type R1 strain. Cellular manganese (Mn ion (Mn2+ analysis showed that Mn2+, copper (Cu2+, and ferric (Fe3+ ions accumulated significantly in the mutant, which suggests that the dr0865 gene is not only involved in the regulation of Mn2+ homeostasis, but also affects the uptake of other ions. In addition, transcriptome profiles under MnCl2 stress showed that the expression of many genes involved in Mn metabolism was significantly different in the wild-type R1 and DR0865 mutant (Mt-0865. Furthermore, we found that the dr0865 gene serves as a positive regulator of the manganese efflux pump gene mntE (dr1236, and as a negative regulator of Mn ABC transporter genes, such as dr2283, dr2284 and dr2523. Therefore, it plays an important role in maintaining the homoeostasis of intracellular Mn (II, and also other Mn2+, zinc (Zn2+ and Cu2+ ions. Based on its role in manganese homeostasis, DR0865 likely belongs to the Mur sub-family of Fur homolog.

  5. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  6. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis

    NARCIS (Netherlands)

    Kamp, Marjon E; Liu, Youtao; Kortholt, Arjan

    2015-01-01

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling

  7. 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.

  8. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins.

    Science.gov (United States)

    Wilson, Rashaun S; Swatek, Kirby N; Thelen, Jay J

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants.

  9. A 60-kilodalton protein component of the counting factor complex regulates group size in Dictyostelium discoideum

    NARCIS (Netherlands)

    Brock, Debra A.; van Egmond, Wouter N.; Shamoo, Yousif; Hatton, R. Diane; Gomer, Richard H.

    Much remains to be understood about how a group of cells or a tissue senses and regulates its size. Dictyostelium discoideum cells sense and regulate the size of groups and fruiting bodies using a secreted 450-kDa complex of proteins called counting factor (CF). Low levels of CF result in large

  10. 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...

  11. Enzymatic mechanisms regulating protein S-nitrosylation: implications in health and disease.

    Science.gov (United States)

    Anand, Puneet; Stamler, Jonathan S

    2012-03-01

    Nitric oxide participates in cellular signal transduction largely through S-nitrosylation of allosteric and active-site cysteine thiols within proteins, forming S-nitroso-proteins (SNO-proteins). S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions, and protein stability. Analogous to other ubiquitous posttranslational modifications that are regulated enzymatically, including phosphorylation and ubiquitinylation, accumulating evidence suggests the existence of enzymatic mechanisms for regulating protein S-nitrosylation. In particular, studies have led to the identification of multiple enzymes (nitrosylases and denitrosylases) that participate in targeted S-nitrosylation or denitrosylation of proteins in physiological settings. Nitrosylases are best characterized in the context of transnitrosylation in which a SNO-protein transfers an NO group to an acceptor protein (Cys-to-Cys transfer), but examples of transnitrosylation catalyzed by metalloproteins (Metal-to-Cys transfer) also exist. By contrast, denitrosylases remove the NO group from SNO-proteins, ultimately using reducing equivalents derived from NADH or NADPH. Here, we focus on the recent discoveries of nitrosylases and denitrosylases and the notion that their aberrant activities may play roles in health and disease.

  12. 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.

  13. 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.

  14. A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened.

    Directory of Open Access Journals (Sweden)

    Seongjin Seo

    2011-11-01

    Full Text Available Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1, interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6 are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.

  15. Testosterone Regulates Tight Junction Proteins and Influences Prostatic Autoimmune Responses

    OpenAIRE

    Meng, Jing; Mostaghel, Elahe A.; Vakar-Lopez, Funda; Montgomery, Bruce; True, Larry; Nelson, Peter S.

    2011-01-01

    Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight jun...

  16. The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS.

    Science.gov (United States)

    Ito, Toshiro; Wellmer, Frank; Yu, Hao; Das, Pradeep; Ito, Natsuko; Alves-Ferreira, Márcio; Riechmann, José Luis; Meyerowitz, Elliot M

    2004-07-15

    The Arabidopsis homeotic gene AGAMOUS (AG) is necessary for the specification of reproductive organs (stamens and carpels) during the early steps of flower development. AG encodes a transcription factor of the MADS-box family that is expressed in stamen and carpel primordia. At later stages of development, AG is expressed in distinct regions of the reproductive organs. This suggests that AG might function during the maturation of stamens and carpels, as well as in their early development. However, the developmental processes that AG might control during organogenesis and the genes that are regulated by this factor are largely unknown. Here we show that microsporogenesis, the process leading to pollen formation, is induced by AG through activation of the SPOROCYTELESS gene (SPL, also known as NOZZLE,NZZ), a regulator of sporogenesis. Furthermore, we demonstrate that SPL can induce microsporogenesis in the absence of AG function, suggesting that AG controls a specific process during organogenesis by activating another regulator that performs a subset of its functions.

  17. Regulation of Saccharomyces cerevisiae DNA polymerase eta transcript and protein.

    Science.gov (United States)

    Pabla, Ritu; Rozario, Donald; Siede, Wolfram

    2008-02-01

    RAD30-encoded DNA polymerase eta functions as a translesion polymerase that can bypass the most frequent types of UV-induced pyrimidine photoproducts in an error-free manner. Although its transcript is UV-inducible in Saccharomyces cerevisiae, Rad30 (studied as a Rad30-Myc fusion) is a stable protein whose levels do not fluctuate following UV treatment or during cell cycle progression. Rad30 protein is subject to monoubiquitination whose level is upregulated in G1 and downregulated during S-phase reentry. This downregulation is accelerated in UV-treated cells. A missense mutation (L577Q) of the ubiquitin binding domain (UBZ) confers a reduced degree of ubiquitination outside of G1 and a complete failure to stably interact with ubiquitinated substrates. This mutation confers a phenotype resembling a complete RAD30 deletion, thus attesting to the significance of the UBZ motif for polymerase eta function in vivo.

  18. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    Arsenic is a metalloid that is toxic to living organisms. The use of arsenic-contaminated ground water for drinking and for irrigation in agriculture presents serious health problems for millions of people in many parts of the world. Arsenate (As(V)) and arsenite (As(III)), the two most widespread...... 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...

  19. Arabidopsis fructokinase-like protein associations are regulated by ATP.

    Science.gov (United States)

    Riggs, John W; Callis, Judy

    2017-05-10

    The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant form in vitro We further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. Protein Kinase C-Regulated Aβ Production and Clearance

    Directory of Open Access Journals (Sweden)

    Taehyun Kim

    2011-01-01

    Full Text Available Alzheimer’s disease (AD is the most common form of dementia among the elderly population. AD, which is characterized as a disease of cognitive deficits, is mainly associated with an increase of amyloid β-peptide (Aβ in the brain. A growing body of recent studies suggests that protein kinase C (PKC promotes the production of the secretory form of amyloid precursor protein (sAPPα via the activation of α-secretase activity, which reduces the accumulation of pathogenic Aβ levels in the brain. Moreover, activation of PKCα and mitogen-activated protein kinase (MAPK is known to increase sAPPα. A novel type of PKC, PKCε, activates the Aβ degrading activity of endothelin converting enzyme type 1 (ECE-1, which might be mediated via the MAPK pathway as well. Furthermore, dysregulation of PKC-MAPK signaling is known to increase Aβ levels in the brain, which results in AD phenotypes. Here, we discuss roles of PKC in Aβ production and clearance and its implication in AD.

  1. Post-transcriptional regulation of ITGB6 protein levels in damaged skeletal muscle

    OpenAIRE

    Ducceschi, Melissa; Clifton, Lisa G.; Stimpson, Stephen A; Billin, Andrew N.

    2014-01-01

    We have identified integrin beta 6 (Itgb6) as a transcript highly enriched in skeletal muscle. This finding is unexpected because Itgb6 is typically associated with epithelial expression domains in normal tissue. Further we find that ITGB6 protein expression in muscle is post-transcriptionally regulated. Uninjured muscle expresses Itgb6 RNA but no ITGB6 protein is detectable. Muscle injury induces ITGB6 protein accumulation rapidly post-injury in myofibers adjacent to the site of injury. As r...

  2. Heterotrimeric G protein-mediated signaling and its non-canonical regulation in the heart.

    Science.gov (United States)

    Zhang, Peng; Kofron, Celinda M; Mende, Ulrike

    2015-05-15

    Heterotrimeric guanine nucleotide-binding proteins (G proteins) regulate a multitude of signaling pathways in mammalian cells by transducing signals from G protein-coupled receptors (GPCRs) to effectors, which in turn regulate cellular function. In the myocardium, G protein signaling occurs in all cardiac cell types and is centrally involved in the regulation of heart rate, pump function, and vascular tone and in the response to hemodynamic stress and injury. Perturbations in G protein-mediated signaling are well known to contribute to cardiac hypertrophy, failure, and arrhythmias. Most of the currently used drugs for cardiac and other diseases target GPCR signaling. In the canonical G protein signaling paradigm, G proteins that are located at the cytoplasmic surface of the plasma membrane become activated after an agonist-induced conformational change of GPCRs, which then allows GTP-bound Gα and free Gβγ subunits to activate or inhibit effector proteins. Research over the past two decades has markedly broadened the original paradigm with a GPCR-G protein-effector at the cell surface at its core by revealing novel binding partners and additional subcellular localizations for heterotrimeric G proteins that facilitate many previously unrecognized functional effects. In this review, we focus on non-canonical and epigenetic-related mechanisms that regulate heterotrimeric G protein expression, activation, and localization and discuss functional consequences using cardiac examples where possible. Mechanisms reviewed involve microRNAs, histone deacetylases, chaperones, alternative modes of G protein activation, and posttranslational modifications. Some of these newly characterized mechanisms may be further developed into novel strategies for the treatment of cardiac disease and beyond. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Long-Timescale Dynamics and Regulation of Sec-Facilitated Protein Translocation

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2012-10-01

    Full Text Available We present a coarse-grained modeling approach that spans the nanosecond- to minute-timescale dynamics of cotranslational protein translocation. The method enables direct simulation of both integral membrane protein topogenesis and transmembrane domain (TM stop-transfer efficiency. Simulations reveal multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrate that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon lateral gate, and it is predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides insight into the molecular regulation of the Sec translocon.

  4. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

    Science.gov (United States)

    Gavi, Shai; Shumay, Elena; Wang, Hsien-yu; Malbon, Craig C

    2006-03-01

    G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

  5. 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

    to have wide-ranging and substantial effects on cellular function, both as part of signalling network modulation and more directly by modifying the function of key proteins. In this study, we show that PTM regulation is differentially targeted at different areas of the proteome, and that cytoskeletal...... proteins involved in neuronal process extension and maintenance are both more heavily modified and more frequently regulated at a PTM level. This suggests a clear role not only for PTMs in these processes, but possibly also for heavy protein modification in general. BIOLOGICAL SIGNIFICANCE: This study...

  6. Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins

    Directory of Open Access Journals (Sweden)

    Stephanie C. Huelga

    2012-02-01

    Full Text Available Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  7. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins.

    Science.gov (United States)

    Huelga, Stephanie C; Vu, Anthony Q; Arnold, Justin D; Liang, Tiffany Y; Liu, Patrick P; Yan, Bernice Y; Donohue, John Paul; Shiue, Lily; Hoon, Shawn; Brenner, Sydney; Ares, Manuel; Yeo, Gene W

    2012-02-23

    Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  8. 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.

  9. BET bromodomain proteins and epigenetic regulation of inflammation: implications for type 2 diabetes and breast cancer.

    Science.gov (United States)

    Nicholas, Dequina A; Andrieu, Guillaume; Strissel, Katherine J; Nikolajczyk, Barbara S; Denis, Gerald V

    2017-01-01

    Chronic inflammation drives pathologies associated with type 2 diabetes (T2D) and breast cancer. Obesity-driven inflammation may explain increased risk and mortality of breast cancer with T2D reported in the epidemiology literature. Therapeutic approaches to target inflammation in both T2D and cancer have so far fallen short of the expected improvements in disease pathogenesis or outcomes. The targeting of epigenetic regulators of cytokine transcription and cytokine signaling offers one promising, untapped approach to treating diseases driven by inflammation. Recent work has deeply implicated the Bromodomain and Extra-Terminal domain (BET) proteins, which are acetylated histone "readers", in epigenetic regulation of inflammation. This review focuses on inflammation associated with T2D and breast cancer, and the possibility of targeting BET proteins as an approach to regulating inflammation in the clinic. Understanding inflammation in the context of BET protein regulation may provide a basis for designing promising therapeutics for T2D and breast cancer.

  10. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  11. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. RNA binding proteins in the regulation of heart development.

    Science.gov (United States)

    Blech-Hermoni, Yotam; Ladd, Andrea N

    2013-11-01

    In vivo, RNA molecules are constantly accompanied by RNA binding proteins (RBPs), which are intimately involved in every step of RNA biology, including transcription, editing, splicing, transport and localization, stability, and translation. RBPs therefore have opportunities to shape gene expression at multiple levels. This capacity is particularly important during development, when dynamic chemical and physical changes give rise to complex organs and tissues. This review discusses RBPs in the context of heart development. Since the targets and functions of most RBPs--in the heart and at large--are not fully understood, this review focuses on the expression and roles of RBPs that have been implicated in specific stages of heart development or developmental pathology. RBPs are involved in nearly every stage of cardiogenesis, including the formation, morphogenesis, and maturation of the heart. A fuller understanding of the roles and substrates of these proteins could ultimately provide attractive targets for the design of therapies for congenital heart defects, cardiovascular disease, or cardiac tissue repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

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    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.

  14. Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control

    Science.gov (United States)

    Han, Sumin; Lone, Museer A.; Schneiter, Roger; Chang, Amy

    2010-01-01

    Yeast members of the ORMDL family of endoplasmic reticulum (ER) membrane proteins play a central role in lipid homeostasis and protein quality control. In the absence of yeast Orm1 and Orm2, accumulation of long chain base, a sphingolipid precursor, suggests dysregulation of sphingolipid synthesis. Physical interaction between Orm1 and Orm2 and serine palmitoyltransferase, responsible for the first committed step in sphingolipid synthesis, further supports a role for the Orm proteins in regulating sphingolipid synthesis. Phospholipid homeostasis is also affected in orm1Δ orm2Δ cells: the cells are inositol auxotrophs with impaired transcriptional regulation of genes encoding phospholipid biosynthesis enzymes. Strikingly, impaired growth of orm1Δ orm2Δ cells is associated with constitutive unfolded protein response, sensitivity to stress, and slow ER-to-Golgi transport. Inhibition of sphingolipid synthesis suppresses orm1Δ orm2Δ phenotypes, including ER stress, suggesting that disrupted sphingolipid homeostasis accounts for pleiotropic phenotypes. Thus, the yeast Orm proteins control membrane biogenesis by coordinating lipid homeostasis with protein quality control. PMID:20212121

  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

    . These include glucose uptake, glycogen synthesis, post-exercise insulin sensitivity, fatty acid (FA) uptake, intramuscular triacylglyceride hydrolysis, FA oxidation, suppression of protein synthesis, proteolysis, autophagy and transcriptional regulation of genes relevant to promoting an oxidative phenotype.......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...

  16. Dexamethasone Regulates Cochlear Expression of Deafness-associated Proteins Myelin Protein Zero and Heat Shock Protein 70, as Revealed by iTRAQ Proteomics.

    Science.gov (United States)

    Maeda, Yukihide; Fukushima, Kunihiro; Kariya, Shin; Orita, Yorihisa; Nishizaki, Kazunori

    2015-08-01

    Using proteomics, we aimed to identify the proteins differentially regulated by dexamethasone in the mouse cochlea based on mass-spectrometry data. Glucocorticoid therapy is widely used for many forms of sensorineural hearing loss; however, the molecular mechanism of its action in the cochlea remains poorly understood. Dexamethasone or control saline was intratympanically applied to the cochleae of mice. Twelve hours after application, proteins differentially regulated by dexamethasone in the cochlea were analyzed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-mass spectrometry. Next, dexamethasone-dependent regulation of these proteins was verified in the cochleae of mice with noise-induced hearing loss (NIHL) and systemic administration of dexamethasone by western blotting. Immunolocalizations of these proteins were examined in cochleae with NIHL. A total of 247 proteins with a greater than 95% confidence interval of protein identification were found, and 11 differentially expressed proteins by dexamethasone were identified by the iTRAQ-mass spectrometry. One protein, myelin protein zero (Mpz), was upregulated (1.870 ± 0.201-fold change, p < 0.01) at 6 hours post-systemic dexamethasone and noise exposure in a mouse model of NIHL. Heat shock protein 70 (Hsp70) was downregulated (0.511 ± 0.274-fold change, p < 0.05) at 12 hours post-systemic dexamethasone. Immunohistochemistry confirmed Mpz localization to the efferent and afferent processes of the spiral neurons, whereas Hsp70 showed a more ubiquitous expression pattern in the cochlea. Both Mpz and Hsp70 have been reported to be closely associated with sensorineural hearing loss in humans. Dexamethasone significantly modulated the expression levels of these proteins in the cochleae of mice.

  17. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Science.gov (United States)

    Zhao, Runzhen; Liang, Xinrong; Zhao, Meimi; Liu, Shan-Lu; Huang, Yao; Idell, Steven; Li, Xiumin; Ji, Hong-Long

    2014-01-01

    Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  19. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Directory of Open Access Journals (Sweden)

    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

  20. Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation.

    Science.gov (United States)

    Okumura, Masaki; Shimamoto, Shigeru; Hidaka, Yuji

    2014-04-01

    Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI. Copyright © 2014 John Wiley & Sons, Inc.

  1. Protein Phosphatase-1 Regulates Expression of Neuregulin-1

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    Tatiana Ammosova

    2016-12-01

    Full Text Available Protein phosphatase 1 (PP1, a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1 and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9. Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1 that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner.

  2. TIS11 Family Proteins and Their Roles in Posttranscriptional Gene Regulation

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    Maria Baou

    2009-01-01

    Full Text Available Posttranscriptional regulation of gene expression of mRNAs containing adenine-uridine rich elements (AREs in their 3 untranslated regions is mediated by a number of different proteins that interact with these elements to either stabilise or destabilise them. The present review concerns the TPA-inducible sequence 11 (TIS11 protein family, a small family of proteins, that appears to interact with ARE-containing mRNAs and promote their degradation. This family of proteins has been extensively studied in the past decade. Studies have focussed on determining their biochemical functions, identifying their target mRNAs, and determining their roles in cell functions and diseases.

  3. Deciphering the protein-protein interaction network regulating hepatocellular carcinoma metastasis.

    Science.gov (United States)

    Qin, Guoxuan; Dang, Mengjiao; Gao, Huajun; Wang, Hao; Luo, Fengting; Chen, Ruibing

    2017-09-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of mortality related to cancer all over the world. To better understand the molecular mechanisms of HCC metastasis, we analyzed the proteome of three HCC cell lines with different metastasis potentials by quantitative proteomics and bioinformatics analysis. As a result, we identified 378 cellular proteins potentially associated to HCC metastasis, and constructed a highly connected protein-protein interaction (PPI) network. Functional annotation of the network uncovered prominent pathways and key roles of these proteins, suggesting that the metabolism and cytoskeleton biological processes are greatly involved with HCC metastasis. Furthermore, the integrative network analysis revealed a rich-club organization within the PPI network, indicating a hub center of connections. The rich-club nodes include several well-known cancer-related proteins, such as proto-oncogene non-receptor tyrosine kinase (SRC) and pyruvate kinase M2 (PKM2). Moreover, the differential expressions of two identified proteins, including PKM2 and actin-related protein 2/3 complex subunit 4 (ARPC4), were validated using Western blotting. These two proteins were revealed as potential prognostic markers for HCC as shown by survival rate analysis. Copyright © 2017. Published by Elsevier B.V.

  4. 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...

  5. Protein phosphatase 1 (PP1 is a post-translational regulator of the mammalian circadian clock.

    Directory of Open Access Journals (Sweden)

    Isabelle Schmutz

    Full Text Available Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1 as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock.

  6. Isolation of nuclear proteins from flax (Linum usitatissimum L. seed coats for gene expression regulation studies

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    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.

  7. Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

    Science.gov (United States)

    Mackenzie, Kimberly D; Lumsden, Amanda L; Guo, Feng; Duffield, Michael D; Chataway, Timothy; Lim, Yoon; Zhou, Xin-Fu; Keating, Damien J

    2016-09-01

    Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling. © 2016 International Society for Neurochemistry.

  8. Wnt proteins regulate acetylcholine receptor clustering in muscle cells.

    Science.gov (United States)

    Zhang, Bin; Liang, Chuan; Bates, Ryan; Yin, Yiming; Xiong, Wen-Cheng; Mei, Lin

    2012-02-06

    The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

  9. Wnt proteins regulate acetylcholine receptor clustering in muscle cells

    Directory of Open Access Journals (Sweden)

    Zhang Bin

    2012-02-01

    Full Text Available Abstract Background The neuromuscular junction (NMJ is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. Results In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16 that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. Conclusions These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

  10. Antiproliferative protein Tob directly regulates c-myc proto-oncogene expression through cytoplasmic polyadenylation element-binding protein CPEB.

    Science.gov (United States)

    Ogami, K; Hosoda, N; Funakoshi, Y; Hoshino, S

    2014-01-02

    The regulation of mRNA deadenylation constitutes a pivotal mechanism of the post-transcriptional control of gene expression. Here we show that the antiproliferative protein Tob, a component of the Caf1-Ccr4 deadenylase complex, is involved in regulating the expression of the proto-oncogene c-myc. The c-myc mRNA contains cis elements (CPEs) in its 3'-untranslated region (3'-UTR), which are recognized by the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB recruits Caf1 deadenylase through interaction with Tob to form a ternary complex, CPEB-Tob-Caf1, and negatively regulates the expression of c-myc by accelerating the deadenylation and decay of its mRNA. In quiescent cells, c-myc mRNA is destabilized by the trans-acting complex (CPEB-Tob-Caf1), while in cells stimulated by the serum, both Tob and Caf1 are released from CPEB, and c-Myc expression is induced early after stimulation by the stabilization of its mRNA as an 'immediate-early gene'. Collectively, these results indicate that Tob is a key factor in the regulation of c-myc gene expression, which is essential for cell growth. Thus, Tob appears to function in the control of cell growth at least, in part, by regulating the expression of c-myc.

  11. Skeletal muscle as a regulator of the longevity protein, Klotho

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    Keith G Avin

    2014-06-01

    Full Text Available Klotho is a powerful longevity protein that has been linked to the prevention of muscle atrophy, osteopenia, and cardiovascular disease. Similar anti-aging effects have also been ascribed to exercise and physical activity. While an association between muscle function and klotho expression has been previously suggested from longitudinal cohort studies, a direct relationship between circulating klotho and skeletal muscle has not been investigated. In this paper, we present a review of the literature and preliminary evidence that, together, suggests klotho expression may be modulated by skeletal muscle activity. Our pilot clinical findings performed in young and aged individuals suggest that circulating klotho levels are upregulated in response to an acute exercise bout, but that the response may be dependent on fitness level. A similar upregulation of circulating klotho is also observed in response to an acute exercise in young and old mice, suggesting this may be a good model for mechanistically probing the role of physical activity on klotho expression. Finally, we highlight overlapping signaling pathways that are modulated by both klotho and skeletal muscle and propose potential mechanisms for cross-talk between the two. It is hoped that this review will stimulate further consideration of the relationship between skeletal muscle activity and klotho expression, potentially leading to important insights into the well-documented systemic anti-aging effects of exercise.

  12. 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.

  13. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development.

    Science.gov (United States)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin R

    2014-04-14

    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 to have wide-ranging and substantial effects on cellular function, both as part of signalling network modulation and more directly by modifying the function of key proteins. In this study, we show that PTM regulation is differentially targeted at different areas of the proteome, and that cytoskeletal proteins involved in neuronal process extension and maintenance are both more heavily modified and more frequently regulated at a PTM level. This suggests a clear role not only for PTMs in these processes, but possibly also for heavy protein modification in general. This study 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 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 information regarding aspects of the PTMome which are not normally examined, and illuminates the role of PTMs on a more detailed, protein-centric and site-specific level in a biological context. The widespread yet uneven distributions observed will be relevant to those readers with an interest in the mechanisms of distribution of PTMS and their functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Seminal plasma proteins regulate the association of lipids and proteins within detergent-resistant membrane domains of bovine spermatozoa.

    Science.gov (United States)

    Girouard, Julie; Frenette, Gilles; Sullivan, Robert

    2008-05-01

    Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.

  15. Solution conformation of the response regulator proteins from Deinococcus radiodurans studied by SAXS

    Science.gov (United States)

    Li, Li-Qin; Liu, Ying; Liu, Peng; Dong, Yu-Hui

    2011-10-01

    In this paper the solution conformation of the response regulator proteins from Deinococcus radiodurans was studied by small-angle X-ray scattering (SAXS). The SAXS curves of Dr-rrA in solutions were obtained at Beamline 1W2A of Beijing Synchrotron Radiation Facility (BSRF). Two possible conformations of the response regulator proteins, compact and incompact conformations, have been represented by the known crystallographic structures. And theoretical solution scattering curves of the two possible conformations were calculated and fitted to the experimental scattering curve of Dr-rrA, respectively. The result indicates that the solution conformation of the response regulator proteins is inclined to the compact one, which is in agreement with the result of biochemical experiments.

  16. Protein Digestion-Derived Peptides and the Peripheral Regulation of Food Intake

    Directory of Open Access Journals (Sweden)

    Benoit Cudennec

    2017-04-01

    Full Text Available The gut plays a central role in energy homeostasis. Food intake regulation strongly relies on the gut–brain axis, and numerous studies have pointed out the significant role played by gut hormones released from enteroendocrine cells. It is well known that digestive products of dietary protein possess a high satiating effect compared to carbohydrates and fat. Nevertheless, the processes occurring in the gut during protein digestion involved in the short-term regulation of food intake are still not totally unraveled. This review provides a concise overview of the current data concerning the implication of food-derived peptides in the peripheral regulation of food intake with a focus on the gut hormones cholecystokinin and glucagon-like peptide 1 regulation and the relationship with some aspects of glucose homeostasis.

  17. Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

    Science.gov (United States)

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

    2012-01-01

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

  18. mTOR Signaling in Protein Translation Regulation: Implications in Cancer Genesis and Therapeutic Interventions

    Directory of Open Access Journals (Sweden)

    Mehvish Showkat

    2014-01-01

    Full Text Available mTOR is a central nutrient sensor that signals a cell to grow and proliferate. Through distinct protein complexes it regulates different levels of available cellular energy substrates required for cell growth. One of the important functions of the complex is to maintain available amino acid pool by regulating protein translation. Dysregulation of mTOR pathway leads to aberrant protein translation which manifests into various pathological states. Our review focuses on the role mTOR signaling plays in protein translation and its physiological role. It also throws some light on available data that show translation dysregulation as a cause of pathological complexities like cancer and the available drugs that target the pathway for cancer treatment.

  19. Heme metabolism in stress regulation and protein production: From Cinderella to a key player.

    Science.gov (United States)

    Martínez, J L; Petranovic, D; Nielsen, J

    2016-04-02

    Heme biosynthesis is a highly conserved pathway which is present in all kingdoms, from Archaea to higher organisms such as plants and mammals. The heme molecule acts as a prosthetic group for different proteins and enzymes involved in energy metabolism and reactions involved in electron transfer. Based on our recent findings and other recent reports, we here illustrate that heme is more than a co-factor. We also discuss the necessity to gain more insight into the heme biosynthesis pathway regulation, as this interacts closely with overall stress control. Understanding heme biosynthesis and its regulation could impact our ability to develop more efficient yeast cell factories for heterologous protein production.

  20. Targeting pH regulating proteins for cancer therapy-Progress and limitations.

    Science.gov (United States)

    Parks, Scott K; Pouysségur, Jacques

    2017-04-01

    Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

    Science.gov (United States)

    Pruett-Miller, Shondra M.; Reading, David W.; Porter, Shaina N.; Porteus, Matthew H.

    2009-01-01

    Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity. PMID:19214211

  2. Overview of OVATE FAMILY PROTEINS, A Novel Class of Plant-Specific Growth Regulators.

    Science.gov (United States)

    Wang, Shucai; Chang, Ying; Ellis, Brian

    2016-01-01

    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.

  3. Protein phosphatase 2A isotypes regulate cell surface expression of the T cell receptor

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

    2001-01-01

    show that inhibition of the serine/threonine protein phosphatase PP2A family had a biphasic effect on TCR expression. Thus, low concentrations of PP2A inhibitors induced TCR down-regulation, whereas higher concentrations of PP2A inhibitors induced TCR up-regulation. The effect of PP2A inhibition...... regulatory role for PP2A in both exocytosis and endocytosis....

  4. 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...... of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes....

  5. Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1

    OpenAIRE

    Zhang, Xiujun; Qu, Yinbo; Qin, Yuqi

    2016-01-01

    Background Heterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromat...

  6. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    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

  7. Roles of Piwi Proteins in Transcriptional Regulation Mediated by HP1s in Cultured Silkworm Cells

    Science.gov (United States)

    Tatsuke, Tsuneyuki; Zhu, Li; Li, Zhiqing; Mitsunobu, Hitoshi; Yoshimura, Kaito; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2014-01-01

    Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Recently, Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a). However, similar interactions have not been reported in other higher eukaryotes. Here we show that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. We found that HP1a plays an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. We also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b. PMID:24637637

  8. Regulator of G-protein signaling - 5 (RGS5 is a novel repressor of hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    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.

  9. Study of the Stn protein in Salmonella; a regulator of membrane composition and integrity.

    Science.gov (United States)

    Nakano, Masayuki; Yamasaki, Eiki; Moss, Joel; Hirayama, Toshiya; Kurazono, Hisao

    2015-01-01

    Our studies were undertaken to develop new insights into the function of the Salmonella Stn protein. An analysis of total cell membrane protein fraction suggested the possibility that Stn associates with OmpA. This possibility was confirmed by immunogold labeling using anti-OmpA antibody and far-western blotting. From these results, we conclude that Stn regulates membrane composition and integrity in Salmonella.

  10. Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications

    OpenAIRE

    Scott, Anthony; Song, Jing; Ewing, Rob; Wang, Zhenghe

    2014-01-01

    DNA methylation is an important epigenetic mechanism that ensures correct gene expression and maintains genetic stability. DNA methyltransferase 1 (DNMT1) is the primary enzyme that maintains DNA methylation during replication. Dysregulation of DNMT1 is implicated in a variety of diseases. DNMT1 protein stability is regulated via various post-translational modifications, such as acetylation and ubiquitination, but also through protein–protein interactions. These mechanisms ensure DNMT1 is pro...

  11. The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function

    OpenAIRE

    Pedrotti, Simona; Giudice, Jimena; Dagnino-Acosta, Adan; Knoblauch, Mark; Singh, Ravi K.; Hanna, Amy; Mo, Qianxing; Hicks, John; Hamilton, Susan; Cooper, Thomas A

    2015-01-01

    The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic ap...

  12. Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

    Science.gov (United States)

    Singhmar, Pooja; Kumar, Arun

    2011-01-01

    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.

  13. Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

    Directory of Open Access Journals (Sweden)

    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.

  14. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

    Directory of Open Access Journals (Sweden)

    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.

  15. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    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.

  16. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    Science.gov (United States)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  17. Dicer-like Proteins Regulate the Growth, Conidiation, and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation.

    Directory of Open Access Journals (Sweden)

    Wenjun Deng

    2015-12-01

    Full Text Available Post-transcriptional modifications of transfer RNAs (tRNAs have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5 and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2 modifications catalyzed by tRNA methyltransferase 9 (Trm9 in tRNAArg(UCU and tRNAGlu(UUC and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

  20. Regulation of RCAN1 Protein Activity by Dyrk1A Protein-mediated Phosphorylation*

    OpenAIRE

    Jung, Min-Su; Park, Jung-Hwa; Ryu, Young Shin; Choi, Sun-Hee; Yoon, Song-Hee; Kwen, Mi-Yang; Oh, Ji Youn; Song, Woo-Joo; Chung, Sul-Hee

    2011-01-01

    Two genes on chromosome 21, namely dual specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) and regulator of calcineurin 1 (RCAN1), have been implicated in some of the phenotypic characteristics of Down syndrome, including the early onset of Alzheimer disease. Although a link between Dyrk1A and RCAN1 and the nuclear factor of activated T cells (NFAT) pathway has been reported, it remains unclear whether Dyrk1A directly interacts with RCAN1. In the present study, Dyrk1A is shown ...

  1. 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

  2. Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

    Science.gov (United States)

    Nozaki, Shohei; Katoh, Yohei; Terada, Masaya; Michisaka, Saki; Funabashi, Teruki; Takahashi, Senye; Kontani, Kenji; Nakayama, Kazuhisa

    2017-02-01

    ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. © 2017. Published by The Company of Biologists Ltd.

  3. Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

    Directory of Open Access Journals (Sweden)

    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.

  4. Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

    Science.gov (United States)

    Criswell, D. S.; Carson, J. A.; Booth, F. W.

    1996-01-01

    Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.

  5. A Dynamic Response Regulator Protein Modulates G-Protein–Dependent Polarity in the Bacterium Myxococcus xanthus

    Science.gov (United States)

    Zhang, Yong; Guzzo, Mathilde; Ducret, Adrien; Li, Yue-Zhong; Mignot, Tâm

    2012-01-01

    Migrating cells employ sophisticated signal transduction systems to respond to their environment and polarize towards attractant sources. Bacterial cells also regulate their polarity dynamically to reverse their direction of movement. In Myxococcus xanthus, a GTP-bound Ras-like G-protein, MglA, activates the motility machineries at the leading cell pole. Reversals are provoked by pole-to-pole switching of MglA, which is under the control of a chemosensory-like signal transduction cascade (Frz). It was previously known that the asymmetric localization of MglA at one cell pole is regulated by MglB, a GTPase Activating Protein (GAP). In this process, MglB specifically localizes at the opposite lagging cell pole and blocks MglA localization at that pole. However, how MglA is targeted to the leading pole and how Frz activity switches the localizations of MglA and MglB synchronously remained unknown. Here, we show that MglA requires RomR, a previously known response regulator protein, to localize to the leading cell pole efficiently. Specifically, RomR-MglA and RomR-MglB complexes are formed and act complementarily to establish the polarity axis, segregating MglA and MglB to opposite cell poles. Finally, we present evidence that Frz signaling may regulate MglA localization through RomR, suggesting that RomR constitutes a link between the Frz-signaling and MglAB polarity modules. Thus, in Myxococcus xanthus, a response regulator protein governs the localization of a small G-protein, adding further insight to the polarization mechanism and suggesting that motility regulation evolved by recruiting and combining existing signaling modules of diverse origins. PMID:22916026

  6. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Science.gov (United States)

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  7. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Stanton Peter G

    2011-05-01

    Full Text Available Abstract Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL 11 regulates human endometrial epithelial cells (hEEC adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2 and flotillin-1 (FLOT1, were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle. Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary h

  8. 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

  9. 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.

  10. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    DEFF Research Database (Denmark)

    Brandauer, Josef; Vienberg, Sara Gry; Andersen, Marianne Agerholm

    2013-01-01

    -activated protein kinase (AMPK) increases sirtuin activity by elevating NAD levels. As NAM directly inhibits sirtuins, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary...... for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependant. One-legged knee-extensor exercise training in humans increased Nampt protein...

  11. 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...

  12. Nerve growth factor selectively regulates expression of transcripts encoding ribosomal proteins

    Directory of Open Access Journals (Sweden)

    Töröcsik Béata

    2002-02-01

    Full Text Available Abstract Background NGF exerts a variety of actions including promotion of neuronal differentiation and survival. The PC12 rat pheochromocytoma cell line has proved valuable for studying how NGF works and has revealed that the NGF mechanism includes regulation of gene expression. Accordingly, we used SAGE (Serial Analysis of Gene Expression to compare levels of specific transcripts in PC12 cells before and after long-term NGF exposure. Of the approximately 22,000 transcripts detected and quantified, 4% are NGF-regulated by 6-fold or more. Here, we used database information to identify transcripts in our SAGE libraries that encode ribosomal proteins and have compared the effect of NGF on their relative levels of expression. Results Among the transcripts detected in our SAGE analysis, 74 were identified as encoding ribosomal proteins. Ribosomal protein transcripts were among the most abundantly expressed and, for naive and NGF-treated PC12 cells, represented 5.2% and 3.5%, respectively, of total transcripts analyzed. Surprisingly, nearly half of ribosomal protein transcripts underwent statistically significant NGF-promoted alterations in relative abundance, with changes of up to 5-fold. Of the changes, approximately 2/3 represented decreases. A time course revealed that the relative abundance of transcripts encoding RPL9 increases within 1 hr of NGF treatment and is maximally elevated by 8 hr. Conclusions These data establish that NGF selectively changes expression of ribosomal protein transcripts. These findings raise potential roles for regulation of ribosomal protein transcripts in NGF-promoted withdrawal from the cell cycle and neuronal differentiation and indicate that regulation of individual ribosomal protein transcripts is cell- and stimulus-specific.

  13. 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

  14. Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR.

    Science.gov (United States)

    Damianov, Andrey; Ying, Yi; Lin, Chia-Ho; Lee, Ji-Ann; Tran, Diana; Vashisht, Ajay A; Bahrami-Samani, Emad; Xing, Yi; Martin, Kelsey C; Wohlschlegel, James A; Black, Douglas L

    2016-04-21

    Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent to Rbfox crosslinking sites in vivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set of exons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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.

  16. Protein quantitative trait locus study in obesity during weight-loss identifies a leptin regulator

    DEFF Research Database (Denmark)

    Carayol, Jérôme; Chabert, Christian; Di Cara, Alessandro

    2017-01-01

    Thousands of genetic variants have been associated with complex traits through genomewide association studies. However, the functional variants or mechanistic consequences remain elusive. Intermediate traits such as gene expression or protein levels are good proxies of the metabolic state...... of an organism. Proteome analysis especially can provide new insights into the molecular mechanisms of complex traits like obesity. The role of genetic variation in determining protein level variation has not been assessed in obesity. To address this, we design a large-scale protein quantitative trait locus (p......QTL) analysis based on a set of 1129 proteins from 494 obese subjects before and after a weight loss intervention. This reveals 55 BMI-associated cis-pQTLs and trans-pQTLs at baseline and 3 trans-pQTLs after the intervention. We provide evidence for distinct genetic mechanisms regulating BMI-associated proteins...

  17. Protein-Carbohydrate Interaction between Sperm and the Egg-Coating Envelope and Its Regulation by Dicalcin, a Xenopus laevis Zona Pellucida Protein-Associated Protein

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    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.

  18. The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown

    NARCIS (Netherlands)

    Vos, J.W.; Pieuchot, L.; Evrard, J.L.; Janski, N.; Bergdoll, M.; Ronde, de D.

    2008-01-01

    The Targeting Protein for Xklp2 (TPX2) is a central regulator of spindle assembly in vertebrate cells. The absence or excess of TPX2 inhibits spindle formation. We have defined a TPX2 signature motif that is present once in vertebrate sequences but twice in plants. Plant TPX2 is predominantly

  19. 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 Impact factor: 4.125, year: 2016

  20. 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...

  1. Clathrin Assembly Regulated by Adaptor Proteins in Coarse-Grained Models

    NARCIS (Netherlands)

    Giani, M.; den Otter, Wouter K.; Briels, Willem J.

    2016-01-01

    The assembly of clathrin triskelia into polyhedral cages during endocytosis is regulated by adaptor proteins (APs). We explore how APs achieve this by developing coarse-grained models for clathrin and AP2, employing a Monte Carlo click interaction, to simulate their collective aggregation behavior.

  2. SOCS Proteins as Regulators of Inflammatory Responses Induced by Bacterial Infections: A Review

    Directory of Open Access Journals (Sweden)

    Skyla A. Duncan

    2017-12-01

    Full Text Available Severe bacterial infections can lead to both acute and chronic inflammatory conditions. Innate immunity is the first defense mechanism employed against invading bacterial pathogens through the recognition of conserved molecular patterns on bacteria by pattern recognition receptors (PRRs, especially the toll-like receptors (TLRs. TLRs recognize distinct pathogen-associated molecular patterns (PAMPs that play a critical role in innate immune responses by inducing the expression of several inflammatory genes. Thus, activation of immune cells is regulated by cytokines that use the Janus kinase/signal transducers and activators of transcription (JAK/STAT signaling pathway and microbial recognition by TLRs. This system is tightly controlled by various endogenous molecules to allow for an appropriately regulated and safe host immune response to infections. Suppressor of cytokine signaling (SOCS family of proteins is one of the central regulators of microbial pathogen-induced signaling of cytokines, principally through the inhibition of the activation of JAK/STAT signaling cascades. This review provides recent knowledge regarding the role of SOCS proteins during bacterial infections, with an emphasis on the mechanisms involved in their induction and regulation of antibacterial immune responses. Furthermore, the implication of SOCS proteins in diverse processes of bacteria to escape host defenses and in the outcome of bacterial infections are discussed, as well as the possibilities offered by these proteins for future targeted antimicrobial therapies.

  3. Pim serine/threonine kinases regulate the stability of Socs-1 protein

    NARCIS (Netherlands)

    Chen, XP; Losman, JA; Cowan, S; Donahue, E; Fay, S; Vuong, BQ; Nawijn, MC; Capece, D; Cohan, VL; Rothman, P

    2002-01-01

    Studies of SOCS-1-deficient mice have implicated Socs-1 in the suppression of JAK-STAT (Janus tyrosine kinase-signal transducers and activators of transcription) signaling and T cell development. It has been suggested that the levels of Socs-1 protein may be regulated through the proteasome pathway.

  4. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

    DEFF Research Database (Denmark)

    Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

    2012-01-01

    The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJ...

  5. 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 ly...

  6. 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.

  7. Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

    DEFF Research Database (Denmark)

    Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro

    2011-01-01

    In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this ......In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear...... those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-¿B pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1...

  8. 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.

  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. Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis*

    Science.gov (United States)

    Zhang, Jie; Lu, Anrui; Kong, Lulu; Zhang, Qiaoli; Ling, Erjun

    2014-01-01

    Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future. PMID:25368323

  11. Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation.

    Science.gov (United States)

    Wolhuter, Kathryn; Whitwell, Harry J; Switzer, Christopher H; Burgoyne, Joseph R; Timms, John F; Eaton, Philip

    2018-02-01

    S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. 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.

  13. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    Science.gov (United States)

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  14. Nervous wreck, an SH3 adaptor protein that interacts with Wsp, regulates synaptic growth in Drosophila.

    Science.gov (United States)

    Coyle, Ian P; Koh, Young-Ho; Lee, Wyan-Ching Mimi; Slind, Jessica; Fergestad, Tim; Littleton, J Troy; Ganetzky, Barry

    2004-02-19

    We describe the isolation and characterization of nwk (nervous wreck), a temperature-sensitive paralytic mutant that causes excessive growth of larval neuromuscular junctions (NMJs), resulting in increased synaptic bouton number and branch formation. Ultrastructurally, mutant boutons have reduced size and fewer active zones, associated with a reduction in synaptic transmission. nwk encodes an FCH and SH3 domain-containing adaptor protein that localizes to the periactive zone of presynaptic terminals and binds to the Drosophila ortholog of Wasp (Wsp), a key regulator of actin polymerization. wsp null mutants display synaptic overgrowth similar to nwk and enhance the nwk morphological phenotype in a dose-dependent manner. Evolutionarily, Nwk belongs to a previously undescribed family of adaptor proteins that includes the human srGAPs, which regulate Rho activity downstream of Robo receptors. We propose that Nwk controls synapse morphology by regulating actin dynamics downstream of growth signals in presynaptic terminals.

  15. 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...

  16. 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.

  17. Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton.

    Science.gov (United States)

    Yuan, Xue; Serra, Rosa A; Yang, Shuying

    2015-01-01

    Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. © 2014 New York Academy of Sciences.

  18. 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 differentiation of white and brown fat cells is controlled by a similar set of transcription factors, including PPARgamma and C/EBPalpha. However, despite many similarities between the two types of fat cells, they carry out essentially opposite functions in vivo, with white adipocytes being...... 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...

  19. Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA-Protein Affinity Technique.

    Science.gov (United States)

    Truong-Bolduc, Que Chi; Hooper, David C

    2018-01-01

    In this chapter, we describe the step-by-step identification of a putative regulator protein and demonstrate the function of this protein as a repressor of the expression of a specific efflux pump, causing resistance to quinolones in Staphylococcus aureus. We show that the knockout gene mutant has an increase in transcript levels of the target efflux pump when compared to that of the S. aureus parental strain RN6390. We provide a detailed protocol that includes the identification of the DNA-binding transcriptional regulatory protein from S. aureus cell extracts using DNA sequences linked to magnetic beads. In addition, we describe the real-time qRT-PCR assays and MIC testing to evaluate the effects of the regulator on S. aureus drug resistance phenotype.

  20. Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1

    Science.gov (United States)

    Chang, Chuang-Rung; Blackstone, Craig

    2017-01-01

    Mitochondria in cells comprise a tubulovesicular network shaped continuously by complementary fission and fusion events. The mammalian Drp1 protein plays a key role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in the balance between these opposing processes can occur rapidly, indicating that modifications to these proteins may regulate mitochondrial membrane dynamics. We highlight posttranslational modifications of the mitochondrial fission protein Drp1, for which these regulatory mechanisms are best characterized. This dynamin-related GTPase undergoes a number of steps to mediate mitochondrial fission, including translocation from cytoplasm to the mitochondrial outer membrane, higher-order assembly into spirals, GTP hydrolysis associated with a conformational change and membrane deformation, and ultimately disassembly. Many of these steps may be influenced by covalent modification of Drp1. We discuss the dynamic nature of Drp1 modifications and how they contribute not only to the normal regulation of mitochondrial division, but also to neuropathologic processes. PMID:20649536

  1. The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

    Science.gov (United States)

    Pedrotti, Simona; Giudice, Jimena; Dagnino-Acosta, Adan; Knoblauch, Mark; Singh, Ravi K; Hanna, Amy; Mo, Qianxing; Hicks, John; Hamilton, Susan; Cooper, Thomas A

    2015-04-15

    The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. 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.

  3. Influence of the ferric uptake regulator (Fur protein on pathogenicity in Pectobacterium carotovorum subsp. brasiliense.

    Directory of Open Access Journals (Sweden)

    Collins Kipngetich Tanui

    Full Text Available Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692, an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs, biofilm formation, exopolysaccharide (EPS production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132, stress response (SodC-superoxide dismutase, plant cell wall degrading enzymes (PrtA and CelV and motility (FlhC and MotA. We conclude that the ferric uptake regulator protein (Fur of Pcb1692 regulates traits that are important to host-pathogens interactions.

  4. The PHD-containing protein EARLY BOLTING IN SHORT DAYS regulates seed dormancy in Arabidopsis.

    Science.gov (United States)

    Narro-Diego, Laura; López-González, Leticia; Jarillo, Jose A; Piñeiro, Manuel

    2017-10-01

    The Arabidopsis protein EARLY BOLTING IN SHORT DAYS (EBS), a plant-specific transcriptional regulator, is involved in the control of flowering time by repressing the floral integrator FT. The EBS protein binds the H3K4me3 histone mark and interacts with histone deacetylases to modulate gene expression. Here, we show that EBS also participates in the regulation of seed dormancy. ebs mutations cause a reduction in seed dormancy, and the concurrent loss of function of the EBS homologue SHORT LIFE (SHL) enhances this dormancy alteration. Transcriptomic analyses in ebs mutant seeds uncovered the misregulation of several regulators of seed dormancy including the MADS box gene AGAMOUS-LIKE67 (AGL67). AGL67 interacts genetically with EBS in seed dormancy regulation, indicating that both loci act in the same pathway. Interestingly, EBS functions independently of the master regulator gene of dormancy DELAY OF GERMINATION 1 (DOG1) and other genes encoding chromatin remodelling factors involved in the control of seed dormancy. Altogether, these data show that EBS is a central repressor of germination during seed dormancy and that SHL acts redundantly with EBS in the control of this developmental process. Our observations suggest that a tightly regulated crosstalk among histone modifications is necessary for a proper control of seed dormancy. © 2017 John Wiley & Sons Ltd.

  5. Protein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling.

    Science.gov (United States)

    Stomberski, Colin T; Hess, Douglas T; Stamler, Jonathan S

    2018-01-10

    Protein S-nitrosylation, the oxidative modification of cysteine by nitric oxide (NO) to form protein S-nitrosothiols (SNOs), mediates redox-based signaling that conveys, in large part, the ubiquitous influence of NO on cellular function. S-nitrosylation regulates protein activity, stability, localization, and protein-protein interactions across myriad physiological processes, and aberrant S-nitrosylation is associated with diverse pathophysiologies. Recent Advances: It is recently recognized that S-nitrosylation endows S-nitroso-protein (SNO-proteins) with S-nitrosylase activity, that is, the potential to trans-S-nitrosylate additional proteins, thereby propagating SNO-based signals, analogous to kinase-mediated signaling cascades. In addition, it is increasingly appreciated that cellular S-nitrosylation is governed by dynamically coupled equilibria between SNO-proteins and low-molecular-weight SNOs, which are controlled by a growing set of enzymatic denitrosylases comprising two main classes (high and low molecular weight). S-nitrosylases and denitrosylases, which together control steady-state SNO levels, may be identified with distinct physiology and pathophysiology ranging from cardiovascular and respiratory disorders to neurodegeneration and cancer. The target specificity of protein S-nitrosylation and the stability and reactivity of protein SNOs are determined substantially by enzymatic machinery comprising highly conserved transnitrosylases and denitrosylases. Understanding the differential functionality of SNO-regulatory enzymes is essential, and is amenable to genetic and pharmacological analyses, read out as perturbation of specific equilibria within the SNO circuitry. The emerging picture of NO biology entails equilibria among potentially thousands of different SNOs, governed by denitrosylases and nitrosylases. Thus, to elucidate the operation and consequences of S-nitrosylation in cellular contexts, studies should consider the roles of SNO-proteins as

  6. 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.

  7. RNA Binding Protein-Mediated Post-Transcriptional Gene Regulation in Medulloblastoma

    Science.gov (United States)

    Bish, Rebecca; Vogel, Christine

    2014-01-01

    Medulloblastoma, the most common malignant brain tumor in children, is a disease whose mechanisms are now beginning to be uncovered by high-throughput studies of somatic mutations, mRNA expression patterns, and epigenetic profiles of patient tumors. One emerging theme from studies that sequenced the tumor genomes of large cohorts of medulloblastoma patients is frequent mutation of RNA binding proteins. Proteins which bind multiple RNA targets can act as master regulators of gene expression at the post-transcriptional level to co-ordinate cellular processes and alter the phenotype of the cell. Identification of the target genes of RNA binding proteins may highlight essential pathways of medulloblastomagenesis that cannot be detected by study of transcriptomics alone. Furthermore, a subset of RNA binding proteins are attractive drug targets. For example, compounds that are under development as anti-viral targets due to their ability to inhibit RNA helicases could also be tested in novel approaches to medulloblastoma therapy by targeting key RNA binding proteins. In this review, we discuss a number of RNA binding proteins, including Musashi1 (MSI1), DEAD (Asp-Glu-Ala-Asp) box helicase 3 X-linked (DDX3X), DDX31, and cell division cycle and apoptosis regulator 1 (CCAR1), which play potentially critical roles in the growth and/or maintenance of medulloblastoma. PMID:24608801

  8. Regulation of protein synthesis by amino acids in muscle of neonates

    Science.gov (United States)

    Suryawan, Agus; Davis, Teresa A.

    2011-01-01

    The marked increase in skeletal muscle mass during the neonatal period is largely due to a high rate of postprandial protein synthesis that is modulated by an enhanced sensitivity to insulin and amino acids. The amino acid signaling pathway leading to the stimulation of protein synthesis has not been fully elucidated. Among the amino acids, leucine is considered to be a principal anabolic agent that regulates protein synthesis. mTORC1, which controls protein synthesis, has been implicated as a target for leucine. Until recently, there have been few studies exploring the role of amino acids in enhancing muscle protein synthesis in vivo. In this review, we discuss amino acid-induced protein synthesis in muscle in the neonate, focusing on current knowledge of the role of amino acids in the activation of mTORC1 leading to mRNA translation. The role of the amino acid transporters, SNAT2, LAT1, and PAT, in the modulation of mTORC1 activation and the role of amino acids in the activation of putative regulators of mTORC1, i.e., raptor, Rheb, MAP4K3, Vps34, and Rag GTPases, are discussed. PMID:21196241

  9. 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

  10. 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

    2017-08-02

    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.

  11. Cinnamon polyphenols regulate S100β, sirtuins, and neuroactive proteins in rat C6 glioma cells.

    Science.gov (United States)

    Qin, Bolin; Panickar, Kiran S; Anderson, Richard A

    2014-02-01

    Increasing evidence suggests that cinnamon has many health benefits when used in herbal medicine and as a dietary ingredient. The aim of this study was to investigate the effects of an aqueous extract of cinnamon, high in type A polyphenols, on molecular targets in rat C6 glioma cells that underlie their protective effects. C6 rat glioma cells were seeded in 35-mm culture dishes or six-well plates, then were incubated with cinnamon polyphenols at doses of 10 and 20 μg/mL for 24 h. The targeting protein expression, secretion, and phosphorylation were evaluated by immunoprecitation/immunoblotting and immunofluorescence imaging. Cinnamon polyphenols significantly enhanced secretion of S100β, a Ca(2+)-binding protein, and increased intracellular S100β expression after 24 h of incubation, in rat C6 glioma cells. Cinnamon polyphenols also enhanced protein levels of sirtuin 1, 2, and 3, deacetylases important in cell survival, and the tumor suppressor protein, p53, and inhibited the inflammatory factors, tumor necrosis factor alpha, and phospho-p65, a subunit of nuclear factor-κβ. Cinnamon polyphenols also up-regulated levels of phospho-p38, extracellular signal-regulated protein and mitogen-activated protein and kinase-activated protein kinases that may be important for prosurvival functions. Our results indicate that the effects of cinnamon polyphenols on upregulating prosurvival proteins, activating mitogen-activated protein kinase pathways, and decreasing proinflammatory cytokines may contribute to their neuroprotective effects. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. BRCA1 proteins regulate growth of ovarian cancer cells by tethering Ubc9.

    Science.gov (United States)

    Qin, Yunlong; Xu, Jingyao; Aysola, Kartik; Oprea, Gabriela; Reddy, Avinash; Matthews, Roland; Okoli, Joel; Cantor, Alan; Grizzle, William E; Partridge, Edward E; Reddy, E Shyam P; Landen, Charles; Rao, Veena N

    2012-01-01

    Mutation in the BRCA1 gene is associated with increased risk for hereditary breast and ovarian cancers. In sporadic ovarian tumors, BRCA1 dysfunction is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein. Our group has previously reported that BRCA1 proteins, unlike K109R and cancer-predisposing mutant C61G BRCA1 proteins, bind the sole SUMO E2-conjugating enzyme Ubc9. In this study, we examined the result of altered Ubc9 binding and knockdown on the sub-cellular localization and growth inhibitory function of BRCA1 proteins in ovarian cancer cells. Using live imaging of YFP, RFP-tagged BRCA1 and BRCA1a proteins, our results show enhanced cytoplasmic localization of K109R and C61G mutant BRCA1 proteins in ES-2, NIHOVCAR3 and UWB 1.289 ovarian cancer cells. Down-regulation of Ubc9 in ovarian cancer cells using Ubc9 siRNA resulted in cytoplasmic localization of BRCA1 and BRCA1a proteins. These mutant BRCA1a proteins were impaired in their capacity to inhibit growth of ES-2 ovarian cancer cells. Several ovarian cancer cells, including a BRCA1-null ovarian cancer cell line, showed higher levels of expression of Ubc9. This is the first study demonstrating the physiological link between loss of Ubc9 binding and loss of growth suppression of disease-associated mutant BRCA1a proteins in ovarian cancer cells. BRCA1, by turning off or on Ubc9 binding, regulates growth of ovarian cancers.

  13. 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

  14. 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.

  15. Pat1 proteins: regulating mRNAs from birth to death?

    Science.gov (United States)

    Standart, Nancy; Marnef, Aline

    2012-08-01

    Abstract The Pat1 protein family has been the subject of several recent extensive investigations of diverse model systems ranging from yeast, flies and worms to man, using a variety of experimental approaches. Although some contradictions remain, the emerging consensus view is that these RNA-binding proteins act in mRNA decay by physically linking deadenylation with decapping and by regulating gene expression as translational repressors. These multiple functions are present in the single invertebrate Pat1 proteins, whereas, in vertebrates, one Pat1 variant represses translation in early development, while a somatic version synthesised in embrogenesis and in adults acts in mRNA decay. At steady state, Pat1 proteins are found enriched in cytoplasmic P(rocessing)-bodies, and related mRNP complexes and granules. Evidence recently obtained from mammalian tissue culture cells shows that Pat1 shuttles in and out of the nucleus, where it localises to nuclear speckles, PML bodies and nucleolar caps, which suggests RNA-related nuclear functions. Less well understood, Pat1 proteins may play additional roles in miRNA silencing and/or biogenesis, as well in the regulation of viral gene expression. Due to the relatively low level of sequence conservation between Pat1 proteins from different species and lacking any discernable motifs, determining their functional domains has proved difficult, as is obtaining a simple unified view of the location of the binding sites of their interacting proteins in all examined species. Questions that remain to be addressed include the following: 1) What are their roles in the nucleus? 2) What is the link, if one exists, between their cytoplasmic and nuclear roles? 3) Do they have specific mRNA targets? 4) Which signalling pathways regulate their P-body localisation in mammalian cells, which may affect quiescent cell survival?

  16. Two distinct mechanisms for actin capping protein regulation--steric and allosteric inhibition.

    Science.gov (United States)

    Takeda, Shuichi; Minakata, Shiho; Koike, Ryotaro; Kawahata, Ichiro; Narita, Akihiro; Kitazawa, Masashi; Ota, Motonori; Yamakuni, Tohru; Maéda, Yuichiro; Nitanai, Yasushi

    2010-07-06

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the

  17. 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.

  18. The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.

    Directory of Open Access Journals (Sweden)

    Bungo Akiyoshi

    Full Text Available The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C(Mif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

  19. 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

  20. Protein oxidation mediated by heme-induced active site conversion specific for heme-regulated transcription factor, iron response regulator.

    Science.gov (United States)

    Kitatsuji, Chihiro; Izumi, Kozue; Nambu, Shusuke; Kurogochi, Masaki; Uchida, Takeshi; Nishimura, Shin-ichiro; Iwai, Kazuhiro; O'Brian, Mark R; Ikeda-Saito, Masao; Ishimori, Koichiro

    2016-01-05

    The Bradyrhizobium japonicum transcriptional regulator Irr (iron response regulator) is a key regulator of the iron homeostasis, which is degraded in response to heme binding via a mechanism that involves oxidative modification of the protein. Here, we show that heme-bound Irr activates O2 to form highly reactive oxygen species (ROS) with the "active site conversion" from heme iron to non-heme iron to degrade itself. In the presence of heme and reductant, the ROS scavenging experiments show that Irr generates H2O2 from O2 as found for other hemoproteins, but H2O2 is less effective in oxidizing the peptide, and further activation of H2O2 is suggested. Interestingly, we find a time-dependent decrease of the intensity of the Soret band and appearance of the characteristic EPR signal at g = 4.3 during the oxidation, showing the heme degradation and the successive formation of a non-heme iron site. Together with the mutational studies, we here propose a novel "two-step self-oxidative modification" mechanism, during which O2 is activated to form H2O2 at the heme regulatory motif (HRM) site and the generated H2O2 is further converted into more reactive species such as ·OH at the non-heme iron site in the His-cluster region formed by the active site conversion.

  1. 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.

  2. Regulators of G protein signaling 12 promotes osteoclastogenesis in bone remodeling and pathological bone loss.

    Science.gov (United States)

    Yuan, X; Cao, J; Liu, T; Li, Y-P; Scannapieco, F; He, X; Oursler, M J; Zhang, X; Vacher, J; Li, C; Olson, D; Yang, S

    2015-12-01

    Regulators of G protein signaling (Rgs) have pivotal roles in controlling various cellular processes, such as cell differentiation. How Rgs proteins regulate osteoclast (OC) differentiation, function and bone homeostasis is poorly understood. It was previously demonstrated that Rgs12, the largest protein in the Rgs family, is predominantly expressed in OCs and regulates OC differentiation in vitro. To further understand the role and mechanism of Rgs12 in OC differentiation and bone diseases in vivo, we created OC-targeted Rgs12 knockout mice by using inducible Mx1-Cre and CD11b-Cre. Deletion of Rgs12 in hematopoietic cells or specifically in OC precursors resulted in increased bone mass with decreased OC numbers. Loss of Rgs12 impaired OC differentiation and function with impaired Ca(2+) oscillations and reduced nuclear factor of activated T cells (NFAT) 2 expression. The introduction of wild-type osteoblasts did not rescue the defective osteoclastogenesis. Ectopic expression of NFAT2 rescued defective OC differentiation in CD11b;Rgs12(fl/fl) cells and promoted normal OC differentiation. Moreover, deletion of Rgs12 significantly inhibited pathological osteoclastogenesis and bone destruction in Rgs12-deficient mice that were subjected to ovariectomy and lipodysaccharide for bone loss. Thus our findings demonstrate that Rgs12 is an important regulator in OC differentiation and function and identify Rgs12 as a potential therapeutic target for osteoporosis and inflammation-induced bone loss.

  3. Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

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    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. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    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.

  6. 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.

  7. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer

    Directory of Open Access Journals (Sweden)

    Joshua J. Thompson

    2018-02-01

    Full Text Available 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.

  8. Crosstalk between the unfolded protein response and pathways that regulate pathogenic development in Ustilago maydis.

    Science.gov (United States)

    Heimel, Kai; Freitag, Johannes; Hampel, Martin; Ast, Julia; Bölker, Michael; Kämper, Jörg

    2013-10-01

    The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy.

  9. Hypothalamic STAT proteins: regulation of somatostatin neurones by growth hormone via STAT5b.

    Science.gov (United States)

    Bennett, E; McGuinness, L; Gevers, E F; Thomas, G B; Robinson, I C A F; Davey, H W; Luckman, S M

    2005-03-01

    Signal transducers and activators of transcription (STATs) are a family of transcription factors linked to class I cytokine receptors. In the present study, we investigated whether their distribution in the hypothalamus reflects the feedback regulation by growth hormone and what role they might play in the functioning of target neurones. We demonstrate that each of the seven known STATs has a distinct distribution in the hypothalamus. Notably, the STAT5 proteins, that are important in growth hormone (GH) and prolactin signalling in peripheral tissues, were expressed in somatostatin neurones of the periventricular nucleus and dopamine neurones of the arcuate nucleus. Because somatostatin neurones are regulated by feedback from circulating GH, we investigated the importance of STAT5 in these neurones. We demonstrate that STAT5b protein expression, similar to somatostatin mRNA, is sexually dimorphic in the periventricular nucleus of rats and mice. Furthermore, chronic infusion of male dwarf rats with GH increased the expression of STAT5b, while a single injection of GH into similar rats induced the phosphorylation of STAT5 proteins. The cellular abundance of somatostatin mRNA in STAT5b-deficient mice was significantly reduced in the periventricular nucleus, effectively reducing the sexually dimorphic expression. These results are consistent with the hypothesis that STAT5 proteins are involved in the feedback regulation of somatostatin neurones by GH, and that these neurones may respond to patterned GH secretion to reinforce sexual dimorphism in the GH axis.

  10. 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.

  11. An SMC-like protein binds and regulates Caenorhabditis elegans condensins.

    Science.gov (United States)

    Chao, Lucy Fang-I; Singh, Meha; Thompson, James; Yates, John R; Hagstrom, Kirsten A

    2017-03-01

    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.

  12. Fragile X Mental Retardation Protein expression in the retina is regulated by light.

    Science.gov (United States)

    Guimarães-Souza, E M; Perche, O; Morgans, C W; Duvoisin, R M; Calaza, K C

    2016-05-01

    Fragile X Mental Retardation Protein (FMRP) is a RNA-binding protein that modulates protein synthesis at the synapse and its function is regulated by glutamate. The retina is the first structure that participates in vision, and uses glutamate to transduce electromagnetic signals from light to electrochemical signals to neurons. FMRP has been previously detected in the retina, but its localization has not been studied yet. In this work, our objectives were to describe the localization of FMRP in the retina, to determine whether different exposure to dark or light stimulus alters FMRP expression in the retina, and to compare the pattern in two different species, the mouse and chick. We found that both FMRP mRNA and protein are expressed in the retina. By immunohistochemistry analysis we found that both mouse and chick present similar FMRP expression localized mainly in both plexiform layers and the inner retina. It was also observed that FMRP is down-regulated by 24 h dark adaptation compared to its expression in the retina of animals that were exposed to light for 1 h after 24 h in the dark. We conclude that FMRP is likely to participate in retinal physiology, since its expression changes with light exposure. In addition, the expression pattern and regulation by light of FMRP seems well conserved since it was similar in both mouse and chick. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Regulation of cell structure and function by actin-binding proteins: villin's perspective.

    Science.gov (United States)

    Khurana, Seema; George, Sudeep P

    2008-06-18

    Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.

  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. Signal-transduction networks and the regulation of muscle protein degradation.

    Science.gov (United States)

    Szewczyk, Nathaniel J; Jacobson, Lewis A

    2005-10-01

    Protein degradation in muscle functions in maintaining normal physiological homeostasis and adapting to new homeostatic states, and is required for muscle wasting or atrophy in various pathological states. The interplay between protein synthesis and degradation to maintain homeostasis is complex and responds to a variety of autocrine and intercellular signals from neuronal inputs, hormones, cytokines, growth factors and other regulatory molecules. The intracellular events that connect extracellular signals to the molecular control of protein degradation are incompletely understood, but likely involve interacting signal-transduction networks rather than isolated pathways. We review some examples of signal-transduction systems that regulate protein degradation, including effectors of proteolysis inducing factor (PIF), insulin and insulin-like growth factor (IGF) and their receptors, and fibroblast growth factor (FGF) and its receptors.

  16. Endothelial function and the regulation of muscle protein anabolism in older adults.

    Science.gov (United States)

    Timmerman, K L; Volpi, E

    2013-12-01

    Sarcopenia, the loss of skeletal muscle mass and function with aging, is a major contributor to frailty and morbidity in older adults. Recent evidence has emerged suggesting that endothelial dysfunction and insulin resistance of muscle protein metabolism may significantly contribute to the development of sarcopenia. In this article we review: 1) recent studies and theories on the regulation of skeletal muscle protein balance in older adults; 2) the link between insulin resistance of muscle protein synthesis and endothelial dysfunction in aging; 3) mechanisms for impaired endothelial responsiveness in aging; and 4) potential treatments that may restore the endothelial responsiveness and muscle protein anabolic sensitivity in older adults. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie

    2004-01-01

    The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin...... as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell......-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface...

  18. The cascade of inflammatory cytokines regulating synthesis of acute phase proteins.

    Science.gov (United States)

    Koj, A; Magielska-Zero, D; Bereta, J; Kurdowska, A; Rokita, H; Gauldie, J

    1988-12-01

    The acute phase cytokines: interleukin 1, tumor necrosis factor alpha (cachectin) and beta (lymphotoxin), hepatocyte stimulating factor and several interferons, all belong to the family of endotoxin-inducible, low molecular weight proteins. Their synthesis in macrophages, fibroblasts, lymphocytes, epithelial and some tumor cells is enhanced by the same cytokines, often in the autocrine manner, and suppressed by dexamethasone. The principal hepatocyte stimulating factor (HSF) regulating synthesis of acute phase proteins is probably identical with IFN-beta 2/BSF-2/IL-6, but other inflammatory cytokines (IL-1, TNF alpha, IFN-gamma) are able to induce distinct sets of acute phase proteins, or to modulate the final response pattern. The effect of hrIFN-gamma on production of acute phase proteins by human hepatoma Hep G2 cells is discussed in detail. It is concluded that the cascades of inflammatory cytokines in different tissues represent amplification and regulatory pathways controlling the development of acute phase response in vivo.

  19. SwissProt search result: AK100530 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100530 J023101H04 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 7e-15 ...

  20. SwissProt search result: AK111828 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111828 J023064H19 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 8e-72 ...

  1. SwissProt search result: AK120648 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120648 J013155K09 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 5e-17 ...

  2. SwissProt search result: AK120059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120059 J013001E13 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 2e-24 ...

  3. SwissProt search result: AK101165 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101165 J033028D10 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 1e-16 ...

  4. SwissProt search result: AK105815 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105815 001-203-C10 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Tim...ing of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 5e-11 ...

  5. SwissProt search result: AK065276 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065276 J013002L12 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 6e-17 ...

  6. SwissProt search result: AK073624 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073624 J033048G07 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 1e-16 ...

  7. SwissProt search result: AK111864 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111864 J033025G23 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 1e-16 ...

  8. SwissProt search result: AK121136 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121136 J023077E22 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 5e-21 ...

  9. SwissProt search result: AK066112 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066112 J013051N02 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 5e-22 ...

  10. SwissProt search result: AK067728 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067728 J013116P12 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 7e-24 ...

  11. SwissProt search result: AK067314 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067314 J013102G21 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 7e-24 ...

  12. SwissProt search result: AK102959 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102959 J033115H14 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 1e-16 ...

  13. SwissProt search result: AK099581 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099581 J013040N16 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 6e-17 ...

  14. SwissProt search result: AK059514 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059514 001-029-B09 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Tim...ing of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 4e-22 ...

  15. SwissProt search result: AK072736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072736 J023139D11 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 8e-11 ...

  16. SwissProt search result: AK111899 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111899 J023034P21 (Q9LKL2) Two-component response regulator-like APRR1 (Pseudo-response regulator 1) (Timi...ng of CAB expression 1) (ABI3-interacting protein 1) APRR1_ARATH 1e-16 ...

  17. Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System

    Directory of Open Access Journals (Sweden)

    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.

  18. Gene regulation by translational inhibition is determined by Dicer partnering proteins.

    Science.gov (United States)

    Reis, Rodrigo S; Hart-Smith, Gene; Eamens, Andrew L; Wilkins, Marc R; Waterhouse, Peter M

    2015-02-09

    MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment(1-4). In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition(1). In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript(5). However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown(6). Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized(7-9), but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic systems.

  19. RNA-binding protein hnRNPLL as a critical regulator of lymphocyte homeostasis and differentiation.

    Science.gov (United States)

    Chang, Xing

    2016-05-01

    RNA-binding proteins orchestrate posttranscriptional regulation of gene expression, such as messenger RNA (mRNA) splicing, RNA stability regulation, and translation regulation. Heterogeneous nuclear RNA-binding proteins (hnRNPs) refer to a collection of unrelated RNA-binding proteins predominantly located in the nucleus (Han et al. Biochem J 2010, 430:379-392). Although canonical functions of hnRNPs are to promote pre-mRNA splicing, they are involved in all the processes of RNA metabolism through recognizing specific cis-elements on RNA (Dreyfuss et al. Annu Rev Biochem 1993, 62:289-321; Huelga et al. Cell Rep 2012, 1:167-178; Krecic and Swanson. Curr Opin Cell Biol 1999, 11:363-371). Heterogeneous nuclear RNA-binding protein L like (hnRNPLL) is a tissue-specific hnRNP, which was identified as a regulator of CD45RA to CD45RO switching during memory T-cell development (Oberdoerffer et al. Science 2008, 321:686-691; Topp et al. RNA 2008, 14:2038-2049; Wu et al. Immunity 2008, 29:863-875). Since then, hnRNPLL has emerged as a critical regulator of lymphocyte homeostasis and terminal differentiation, controlling alternative splicing or expression of critical genes for the lymphocytes development (Wu et al. Immunity 2008, 29:863-875; Chang et al. Proc Natl Acad Sci USA 2015, 112:E1888-E1897). This review will summarize recent advances in understanding the functions of hnRNPLL, focusing on its biochemical functions and physiological roles in lymphocyte differentiation and homeostasis. WIREs RNA 2016, 7:295-302. doi: 10.1002/wrna.1335 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  20. 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.

  1. Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

    Science.gov (United States)

    Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

    2015-07-15

    Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  2. A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization.

    Directory of Open Access Journals (Sweden)

    Po-An Yeh

    Full Text Available Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation.

  3. A novel function of twins, B subunit of protein phosphatase 2A, in regulating actin polymerization

    Science.gov (United States)

    Chang, Ching-Jin

    2017-01-01

    Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. PMID:28977036

  4. 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

  5. Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study.

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    Andrés Fernández Parguiña

    Full Text Available BACKGROUND: Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS. Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode. METHODOLOGY/PRINCIPAL FINDINGS: We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS. Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28 and 6 months after the acute event (5. Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbβ3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets. CONCLUSIONS/SIGNIFICANCE: The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets

  6. Protein Kinase C Overactivity Impairs Prefrontal Cortical Regulation of Working Memory

    Science.gov (United States)

    Birnbaum, S. G.; Yuan, P. X.; Wang, M.; Vijayraghavan, S.; Bloom, A. K.; Davis, D. J.; Gobeske, K. T.; Sweatt, J. D.; Manji, H. K.; Arnsten, A. F. T.

    2004-10-01

    The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.

  7. The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila.

    Science.gov (United States)

    Gailite, Ieva; Egger-Adam, Diane; Wodarz, Andreas

    2012-02-01

    Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.

  8. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins.

    Science.gov (United States)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N; Patil, Navinkumar J; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-28

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

  9. 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.

  10. Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

    Directory of Open Access Journals (Sweden)

    Jae-Young Lee

    Full Text Available G9a is a lysine methyltransferase (KMTase for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+ breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+ breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(- breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.

  11. 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 MAPK1-interacting and spindle-stabilizing (MISS)-like (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 secreted alkaline phosphatase (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.

  12. MicroRNA Regulation of Endothelial Junction Proteins and Clinical Consequence

    Directory of Open Access Journals (Sweden)

    Yugang Zhuang

    2016-01-01

    Full Text Available Cellular junctions play a critical role in structural connection and signal communication between cells in various tissues. Although there are structural and functional varieties, cellular junctions include tight junctions, adherens junctions, focal adhesion junctions, and tissue specific junctions such as PECAM-1 junctions in endothelial cells (EC, desmosomes in epithelial cells, and hemidesmosomes in EC. Cellular junction dysfunction and deterioration are indicative of clinical diseases. MicroRNAs (miRNA are ~20 nucleotide, noncoding RNAs that play an important role in posttranscriptional regulation for almost all genes. Unsurprisingly, miRNAs regulate junction protein gene expression and control junction structure integrity. In contrast, abnormal miRNA regulation of junction protein gene expression results in abnormal junction structure, causing related diseases. The major components of tight junctions include zonula occluden-1 (ZO-1, claudin-1, claudin-5, and occludin. The miRNA regulation of ZO-1 has been intensively investigated. ZO-1 and other tight junction proteins such as claudin-5 and occludin were positively regulated by miR-126, miR-107, and miR21 in different models. In contrast, ZO-1, claudin-5, and occludin were negatively regulated by miR-181a, miR-98, and miR150. Abnormal tight junction miRNA regulation accompanies cerebral middle artery ischemia, brain trauma, glioma metastasis, and so forth. The major components of adherens junctions include VE-cadherin, β-catenin, plakoglobin, P120, and vinculin. VE-cadherin and β-catenin were regulated by miR-9, miR-99b, miR-181a, and so forth. These regulations directly affect VE-cadherin-β-catenin complex stability and further affect embryo and tumor angiogenesis, vascular development, and so forth. miR-155 and miR-126 have been shown to regulate PECAM-1 and affect neutrophil rolling and EC junction integrity. In focal adhesion junctions, the major components are integrin β4, paxillin

  13. 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.

  14. WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

    Directory of Open Access Journals (Sweden)

    Aditya Banerjee

    2015-01-01

    Full Text Available 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.

  15. 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.

  16. Effects of energy deficit, dietary protein, and feeding on intracellular regulators of skeletal muscle proteolysis.

    Science.gov (United States)

    Carbone, John W; Margolis, Lee M; McClung, James P; Cao, Jay J; Murphy, Nancy E; Sauter, Edward R; Combs, Gerald F; Young, Andrew J; Pasiakos, Stefan M

    2013-12-01

    This study was undertaken to characterize the ubiquitin proteasome system (UPS) response to varied dietary protein intake, energy deficit (ED), and consumption of a mixed meal. A randomized, controlled trial of 39 adults consuming protein at 0.8 (recommended dietary allowance [RDA]), 1.6 (2×-RDA), or 2.4 (3×-RDA) g · kg(-1) · d(-1) for 31 d. A 10-d weight maintenance (WM) period was followed by 21 d of 40% ED. Ubiquitin (Ub)-mediated proteolysis and associated gene expression were assessed in the postabsorptive (fasted) and postprandial (fed; 480 kcal, 20 g protein) states after WM and ED by using muscle biopsies, fluorescence-based assays, immunoblot analysis, and real-time qRT-PCR. In the assessment of UPS responses to varied protein intakes, ED, and feeding, the RDA, WM, and fasted measures served as appropriate controls. ED resulted in the up-regulation of UPS-associated gene expression, as mRNA expression of the atrogenes muscle RING finger-1 (MuRF1) and atrogin-1 were 1.2- and 1.3-fold higher (Pregardless of dietary protein and energy manipulations. Independent of habitual protein intake and despite increased MuRF1 and atrogin-1 mRNA expression during ED, consuming a protein-containing mixed meal attenuates Ub-mediated proteolysis.

  17. WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

    Science.gov (United States)

    Banerjee, Aditya

    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. PMID:25879071

  18. Mathematical simulation of p53-Mdm2 protein biological system regulation.

    Science.gov (United States)

    Voropaeva, O F; Shokin, Yu I; Nepomnyashchikh, L M; Senchukova, S R

    2014-08-01

    Digital simulation of disorders in the p53 and Mdm2 protein system, involved in repair of DNA defects and in many vital processes, including tumor formation, neurodegeneration, and aging, was carried out on the base of functional nonlinear octoparametric mathematical model. Stress situations were studied, associated with emergence of imbalance in p53 and Mdm2 generation and degradation rates and with disorders in the mechanism of protein interactions, regulated within the framework of the suggested model via dissociation constants. Variants of the analyzed disorders compensation by repeated stress for the system parameters were studied numerically.

  19. Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution.

    Directory of Open Access Journals (Sweden)

    Sarah L Maguire

    2014-01-01

    Full Text Available In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs, which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1 and C. albicans (Cph2 have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1 and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

  20. The effect of natural whey proteins on mechanisms of blood pressure regulation

    Directory of Open Access Journals (Sweden)

    Halina Car

    2014-02-01

    Full Text Available Whey is a rich natural source of peptides and amino acids. It has been reported in numerous studies that biological active peptides isolated from cow’s milk whey may affect blood pressure regulation. Studies on animals and humans have shown that α-lactalbumin and β-lactoglobulin obtained from enzymatically hydrolysed whey inhibit angiotensin converting enzyme (ACE, while lactorphins lower blood pressure by normalizing endothelial function or by opioid receptors dependent mechanism. Whey proteins or their bioactive fragments decrease total cholesterol, LDL fraction and triglycerides, thus reducing the risk factors of cardiovascular diseases. The aim of this review is to discuss the effects of whey proteins on the mechanisms of blood pressure regulation.

  1. Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST.

    Science.gov (United States)

    Nie, Hongyun; Liu, Yueyue; Yin, Xiaolei; Cao, Huiren; Wang, Yanfei; Xiong, Wei; Lin, Yushuang; Xu, Zhigang

    2016-01-01

    Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

  2. MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions.

    Science.gov (United States)

    Cichon, Christoph; Sabharwal, Harshana; Rüter, Christian; Schmidt, M Alexander

    2014-01-01

    Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the 'apical junctional complex-AJC' with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the 'Junctional Adhesion Molecules' (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

  3. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

    Science.gov (United States)

    Gerhardt, Christoph; Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-07-06

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. © 2015 Gerhardt et al.

  4. Environmental stress-mediated changes in transcriptional and translational regulation of protein synthesis in crop plants

    Energy Technology Data Exchange (ETDEWEB)

    Key, J.L.; Nagao, R.T.

    1991-06-01

    The major research activities accomplished during the renewal period focused on defining regulatory mechanisms operative in the heat shock (HS) response and assessing the mechanism of HS-induced thermotolerance in Arabidopsis. HS gene regulation was studied by transcriptional run-off assays, and self regulation of heat shock protein (hsp) levels and synthesis was studied through use of amino acid analogs and protein synthesis inhibitors. Also studied were subcellular localization of hsps during HS and HS recovery, characterization of membrane bound hsps and their effects on membrane proton transport, and the influence of over- or underexpression of HS mRNAs and hsps plant phenotype using transgenic plants. In addition, hsp70 and hsp83 cDNAs/genes are being characterized and their expression patterns will be evaluated. 10 refs. (MHB)

  5. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

    Science.gov (United States)

    Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

    2017-11-01

    Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

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    Jingjing Liang

    2017-01-01

    Full Text Available Ebola (EBOV and Marburg (MARV viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3, a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs, as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA. Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.

  7. Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk.

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    Cathy Slack

    2010-03-01

    Full Text Available Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF-1 signaling (IIS pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the

  8. Expression and regulation of cornified envelope proteins in human corneal epithelium.

    Science.gov (United States)

    Tong, Louis; Corrales, Rosa M; Chen, Zhuo; Villarreal, Arturo L; De Paiva, Cintia S; Beuerman, Roger; Li, De-Quan; Pflugfelder, Stephen C

    2006-05-01

    Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression. Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription-polymerase chain reaction. In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts. Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may

  9. Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

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    Mickey Pentecost

    2015-03-01

    Full Text Available The paramyxovirus matrix (M protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp, a leucine-rich nuclear export signal (NES, and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine. To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated

  10. Neuronal RING finger protein 11 (RNF11 regulates canonical NF-κB signaling

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    Pranski Elaine L

    2012-04-01

    Full Text Available Abstract Background The RING domain-containing protein RING finger protein 11 (RNF11 is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. Methods and results Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11’s association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and

  11. Striatal-enriched protein tyrosine phosphatase regulates the PTPα/Fyn signaling pathway.

    Science.gov (United States)

    Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J

    2015-08-01

    The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61

  12. Predicting network modules of cell cycle regulators using relative protein abundance statistics.

    Science.gov (United States)

    Oguz, Cihan; Watson, Layne T; Baumann, William T; Tyson, John J

    2017-02-28

    Parameter estimation in systems biology is typically done by enforcing experimental observations through an objective function as the parameter space of a model is explored by numerical simulations. Past studies have shown that one usually finds a set of "feasible" parameter vectors that fit the available experimental data equally well, and that these alternative vectors can make different predictions under novel experimental conditions. In this study, we characterize the feasible region of a complex model of the budding yeast cell cycle under a large set of discrete experimental constraints in order to test whether the statistical features of relative protein abundance predictions are influenced by the topology of the cell cycle regulatory network. Using differential evolution, we generate an ensemble of feasible parameter vectors that reproduce the phenotypes (viable or inviable) of wild-type yeast cells and 110 mutant strains. We use this ensemble to predict the phenotypes of 129 mutant strains for which experimental data is not available. We identify 86 novel mutants that are predicted to be viable and then rank the cell cycle proteins in terms of their contributions to cumulative variability of relative protein abundance predictions. Proteins involved in "regulation of cell size" and "regulation of G1/S transition" contribute most to predictive variability, whereas proteins involved in "positive regulation of transcription involved in exit from mitosis," "mitotic spindle assembly checkpoint" and "negative regulation of cyclin-dependent protein kinase by cyclin degradation" contribute the least. These results suggest that the statistics of these predictions may be generating patterns specific to individual network modules (START, S/G2/M, and EXIT). To test this hypothesis, we develop random forest models for predicting the network modules of cell cycle regulators using relative abundance statistics as model inputs. Predictive performance is assessed by the

  13. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans

    OpenAIRE

    Su Yang; Hong Xu; Jiali Wang; Chengzhi Liu; Huizhi Lu; Mengjia Liu; Ye Zhao; Bing Tian; Liangyan Wang; Yuejin Hua

    2016-01-01

    The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in ...

  14. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

    Science.gov (United States)

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

    2015-06-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. Copyright © 2015 the American Physiological Society.

  15. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport.

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    Mariah Rayl

    Full Text Available Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES. The penta-EF-hand (PEF protein apoptosis-linked gene 2 (ALG-2 stabilizes sec31A at ER exit sites (ERES and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport-peflin is a negative regulator of transport.

  16. Expression and Regulation of PIWIL-Proteins and PIWI-Interacting RNAs in Rheumatoid Arthritis.

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    Lenka Pleštilová

    Full Text Available The PIWIL (P-element induced wimpy testis like protein subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA.The expression of PIWIL genes in RA and osteoarthritis (OA synovial tissues and synovial fibroblasts (SF was analysed by Real-time PCR, immunofluorescence and Western blot. The expression of piRNAs was quantified by next generation small RNA sequencing (NGS. The regulation of PIWI/piRNAs, proliferation and methylation of LINE-1 after silencing of PIWIL genes were studied.PIWIL2 and 4 mRNA were similarly expressed in synovial tissues and SF from RA and OA patients. However, on the protein level only PIWIL4 was strongly expressed in SF. Using NGS up to 300 piRNAs were identified in all SF without significant differences in expression levels between RA and OASF. Of interest, the analysis of the co-expression of the detected piRNAs revealed a less tightly regulated pattern of piRNA-823, -4153 and -16659 expression in RASF. In RASF and OASF, stimulation with TNFα+IL1β/TLR-ligands further significantly increased the expression levels of PIWIL2 and 4 mRNA and piRNA-16659 was significantly (4-fold induced upon Poly(I:C stimulation. Silencing of PIWIL2/4 neither affect LINE-1 methylation/expression nor proliferation of RASF.We detected a new class of small regulatory RNAs (piRNAs and their specific binding partners (PIWIL2/4 in synovial fibroblasts. The differential regulation of co-expression of piRNAs in RASF and the induction of piRNA/Piwi-proteins by innate immune stimulators suggest a role in inflammatory processes.

  17. Cryptococcus neoformans Mediator Protein Ssn8 Negatively Regulates Diverse Physiological Processes and Is Required for Virulence

    OpenAIRE

    Lin-Ing Wang; Yu-Sheng Lin; Kung-Hung Liu; Jong, Ambrose Y.; Wei-Chiang Shen

    2011-01-01

    Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed t...

  18. The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly

    OpenAIRE

    Buvelot, Stéphanie; Tatsutani, Sean Y.; Vermaak, Danielle; Biggins, Sue

    2003-01-01

    Ipl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1–GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone ...

  19. Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression

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    Yeates Todd O

    2009-12-01

    Full Text Available Abstract Background Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships. Results We detect coordinated regulation of multiple protein complexes using logic analysis of gene expression data. Specifically, we identify gene triplets composed of genes whose expression profiles are found to be related by various types of logic functions. In order to focus on complexes, we associate the members of a gene triplet with the distinct protein complexes to which they belong. In this way, we identify complexes related by specific kinds of regulatory relationships. For example, we may find that the transcription of complex C is increased only if the transcription of both complex A AND complex B is repressed. We identify hundreds of examples of coordinated regulation among complexes under various stress conditions. Many of these examples involve the ribosome. Some of our examples have been previously identified in the literature, while others are novel. One notable example is the relationship between the transcription of the ribosome, RNA polymerase and mannosyltransferase II, which is involved in N-linked glycan processing in the Golgi. Conclusions The analysis proposed here focuses on relationships among triplets of genes that are not evident when genes are examined in a pairwise fashion as in typical clustering methods. By grouping gene triplets, we are able to decipher coordinated regulation among sets of three complexes. Moreover, using all triplets that involve coordinated regulation with the ribosome

  20. The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

    Science.gov (United States)

    Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

    2014-09-01

    Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

  1. The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

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    Kyriakidis Dimitrios A

    2004-06-01

    Full Text Available Abstract This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the

  2. Type 2C protein phosphatase ABI1 is a negative regulator of strawberry fruit ripening.

    Science.gov (United States)

    Jia, Hai-Feng; Lu, Dong; Sun, Jing-Hua; Li, Chun-Li; Xing, Yu; Qin, Ling; Shen, Yuan-Yue

    2013-04-01

    Although a great deal of progress has been made toward understanding the role of abscisic acid (ABA) in fruit ripening, many components in the ABA signalling pathway remain to be elucidated. Here, a strawberry gene homologous to the Arabidopsis gene ABI1, named FaABI1, was isolated and characterized. The 1641bp cDNA includes an intact open reading frame that encodes a deduced protein of 546 amino acids, in which putative conserved domains were determined by homology analysis. Transcriptional analysis showed that the levels of FaABI1 mRNA expression declined rapidly during strawberry fruit development as evidenced by real-time PCR, semi-quantitative reverse transcription-PCR, and northern blotting analyses, suggesting that the Ser/Thr protein phosphatase PP2C1 encoded by FaABI1 may be involved in fruit ripening as a negative regulator. The results of Tobacco rattle virus-induced gene silencing and PBI121 vector-mediated overexpression suggested that the down- and up-regulation of FaABI1 mRNA expression levels in degreening strawberry fruit could promote and inhibit ripening, respectively. Furthermore, alteration of FaABI1 expression could differentially regulate the transcripts of a set of both ABA-responsive and ripening-related genes, including ABI3, ABI4, ABI5, SnRK2, ABRE1, CHS, PG1, PL, CHI, F3H, DFR, ANS, and UFGT. Taken together, the data provide new evidence for an important role for ABA in regulating strawberry fruit ripening in the processes of which the type 2C protein phosphatase ABI1 serves as a negative regulator. Finally, a possible core mechanism underlying ABA perception and signalling transduction in strawberry fruit ripening is discussed.

  3. Regulation of Fanconi anemia protein FANCD2 monoubiquitination by miR-302

    Energy Technology Data Exchange (ETDEWEB)

    Suresh, Bharathi [Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); College of Medicine, Hanyang University, Seoul (Korea, Republic of); Kumar, A. Madhan [Center of Research Excellence in Corrosion, Research Institute King Fahd University of Petroleum and Minerals, Dhahran (Saudi Arabia); Jeong, Hoe-Su [Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Cho, Youl-Hee [College of Medicine, Hanyang University, Seoul (Korea, Republic of); Ramakrishna, Suresh, E-mail: suresh.ramakris@gmail.com [Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); College of Medicine, Hanyang University, Seoul (Korea, Republic of); Kim, Kye-Seong, E-mail: ks66kim@hanyang.ac.kr [Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); College of Medicine, Hanyang University, Seoul (Korea, Republic of)

    2015-10-16

    Fanconi anemia (FA) is a recessively inherited multigene disease characterized by congenital defects, progressive bone marrow failure, and heightened cancer susceptibility. Monoubiquitination of the FA pathway member FANCD2 contributes to the repair of replication stalling DNA lesions. However, cellular regulation of FANCD2 monoubiquitination remains poorly understood. In the present study, we identified the miR-302 cluster as a potential regulator of FANCD2 by bioinformatics analysis. MicroRNAs (miRNAs) are the major posttranscriptional regulators of a wide variety of biological processes, and have been implicated in a number of diseases. Expression of the exogenous miR-302 cluster (without miR-367) reduced FANCD2 monoubiquitination and nuclear foci formation. Furthermore, miR-302 cells showed extensive chromosomal breakage upon MMC treatment when compared to mock control cells. Taken together, our results suggest that overexpression of miR-302 plays a critical role in the regulation of FANCD2 monoubiquitination, resulting in characteristic defects in DNA repair within cells. - Highlights: • miR-302 binds to the 3′UTR promoter of the FANCD2 gene to regulate gene expression. • miR-302 cluster down-regulates FANCD2 protein expression. • miR-302 cluster reduces FANCD2 monoubiquitination and nuclear foci formation. • miR-302 exhibits the characteristic defects in DNA repair in cells.

  4. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

    Science.gov (United States)

    Peel, Nina; Iyer, Jyoti; Naik, Anar; Dougherty, Michael P; Decker, Markus; O'Connell, Kevin F

    2017-01-01

    In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

  5. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

    Directory of Open Access Journals (Sweden)

    Nina Peel

    2017-01-01

    Full Text Available In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

  6. TDP-43, an ALS linked protein, regulates fat deposition and glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Nancy R Stallings

    Full Text Available The identification of proteins which determine fat and lean body mass composition is critical to better understanding and treating human obesity. TDP-43 is a well-conserved RNA-binding protein known to regulate alternative splicing and recently implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS. While TDP-43 knockout mice show early embryonic lethality, post-natal conditional knockout mice show weight loss, fat depletion, and rapid death, suggesting an important role for TDP-43 in regulating energy metabolism. Here we report, that over-expression of TDP-43 in transgenic mice can result in a phenotype characterized by increased fat deposition and adipocyte hypertrophy. In addition, TDP-43 over-expression in skeletal muscle results in increased steady state levels of Tbc1d1, a RAB-GTPase activating protein involved in Glucose 4 transporter (Glut4 translocation. Skeletal muscle fibers isolated from TDP-43 transgenic mice show altered Glut4 translocation in response to insulin and impaired insulin mediated glucose uptake. These results indicate that levels of TDP-43 regulate body fat composition and glucose homeostasis in vivo.

  7. The level of major urinary proteins is socially regulated in wild Mus musculus musculus.

    Science.gov (United States)

    Janotova, Katerina; Stopka, Pavel

    2011-06-01

    Major urinary proteins (MUPs) are highly polymorphic proteins that have been shown to perform several important functions in the chemical communication of the house mouse, Mus musculus. Production of these proteins in C57Bl/6 females is cyclic, reaching the maximum just before the beginning of estrus. Social environment is an important factor that increases MUP production in both sexes. We examined responsiveness of MUP production to social stimuli in wild mice, Mus musculus musculus. The direction of change of MUP production in males depended on the sex of the stimulus animal. Males up-regulated MUP production when caged with a female, but down-regulated MUP production when caged with a male. Down-regulation was more pronounced in males that were defeated in a male-male encounter. Females responded to a male's presence with a decrease in MUP production. We conclude that social modulation of MUP production is specific and, in coordination with other mechanisms, facilitates adjustment of the animal's odor profile to different social contexts. Our results also suggest that in males, MUPs may play an important role in advertizing the male's quality to females. Furthermore, we highlight the importance of analyzing data corrected with creatinine, which show MUP production on the (post)translational level as well as raw data (non-corrected with creatinine), which represent actual concentrations of MUPs in the urine.

  8. In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response.

    Science.gov (United States)

    Rebnegger, Corinna; Graf, Alexandra B; Valli, Minoska; Steiger, Matthias G; Gasser, Brigitte; Maurer, Michael; Mattanovich, Diethard

    2014-04-01

    Protein production in yeasts is related to the specific growth rate μ. To elucidate on this correlation, we studied the transcriptome of Pichia pastoris at different specific growth rates by cultivating a strain secreting human serum albumin at μ = 0.015 to 0.15 h(-1) in glucose-limited chemostats. Genome-wide regulation revealed that translation-related as well as mitochondrial genes were upregulated with increasing μ, while autophagy and other proteolytic processes, carbon source-responsive genes and other targets of the TOR pathway as well as many transcriptional regulators were downregulated at higher μ. Mating and sporulation genes were most active at intermediate μ of 0.05 and 0.075 h(-1) . At very slow growth (μ = 0.015 h(-1) ) gene regulation differs significantly, affecting many transporters and glucose sensing. Analysis of a subset of genes related to protein folding and secretion reveals that unfolded protein response targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones are upregulated with increasing growth rate while proteolytic degradation of secretory proteins is downregulated. We conclude that a high μ positively affects specific protein secretion rates by acting on multiple cellular processes. © 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made.

  9. Functional analysis of insect molting fluid proteins on the protection and regulation of ecdysis.

    Science.gov (United States)

    Zhang, Jie; Lu, Anrui; Kong, Lulu; Zhang, Qiaoli; Ling, Erjun

    2014-12-26

    Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

    Science.gov (United States)

    Kawabe, Harunori; Ohtani, Misato; Kurata, Tetsuya; Sakamoto, Tomoaki; Demura, Taku

    2018-01-01

    Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

    Science.gov (United States)

    Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

  13. Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana

    Science.gov (United States)

    Cordoba, Elizabeth; Aceves-Zamudio, Denise Lizeth; Hernández-Bernal, Alma Fabiola; Ramos-Vega, Maricela; León, Patricia

    2015-01-01

    Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. PMID:25281700

  14. Transcription factors from Sox family regulate expression of zebrafish Gla-rich protein 2 gene.

    Science.gov (United States)

    Fazenda, C; Conceição, N; Cancela, M L

    2015-11-01

    GRP is a vitamin K-dependent protein with orthologs in all vertebrate taxonomic groups and two paralogs in teleosts. However, no data is available about GRP transcriptional gene regulation. We report a functional promoter for zebrafish grp2 gene regulated by Sox9b, Sox10, Ets1 and Mef2ca as determined by in vitro assays. This was confirmed in vivo for Sox9b and Sox10. Due to the high conservation between human GRP and grp2, its zebrafish ortholog, our results are relevant for the study of human GRP gene regulation and provide new insights towards understanding GRP function. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Kinesin superfamily proteins and the regulation of microtubule dynamics in morphogenesis.

    Science.gov (United States)

    Niwa, Shinsuke

    2015-01-01

    Kinesin superfamily proteins (KIFs) are microtubule-dependent molecular motors that serve as sources of force for intracellular transport and cell division. Recent studies have revealed new roles of KIFs as microtubule stabilizers and depolymerizers, and these activities are fundamental to cellular morphogenesis and mammalian development. KIF2A and KIF19A have microtubule-depolymerizing activities and regulate the neuronal morphology and cilia length, respectively. KIF21A and KIF26A work as microtubule stabilizers that regulate axonal morphology. Morphological defects that are similar to human diseases are observed in mice in which these KIF genes have been deleted. Actually, KIF2A and KIF21A have been identified as causes of human neuronal diseases. In this review, the functions of these atypical KIFs that regulate microtubule dynamics are discussed. Moreover, some interesting unanswered questions and hypothetical answers to them are discussed.

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

    KAUST Repository

    Ruchti, E.

    2016-10-08

    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 neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

  17. Cryptococcus neoformans mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence.

    Directory of Open Access Journals (Sweden)

    Lin-Ing Wang

    Full Text Available Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.

  18. Cryptococcus neoformans mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence.

    Science.gov (United States)

    Wang, Lin-Ing; Lin, Yu-Sheng; Liu, Kung-Hung; Jong, Ambrose Y; Shen, Wei-Chiang

    2011-04-29

    Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.

  19. STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

    Science.gov (United States)

    Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

    2015-01-01

    The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

  20. Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

    Directory of Open Access Journals (Sweden)

    Revati eWani

    2014-10-01

    Full Text Available The perception of reactive oxygen species (ROS has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g. cancer. New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically-distinct alterations to the protein (e.g. sulfenic/sulfinic/sulfonic acid, disulfides. These post-translational modifications (PTM are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

  1. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  2. Synthetic zinc finger proteins: the advent of targeted gene regulation and genome modification technologies.

    Science.gov (United States)

    Gersbach, Charles A; Gaj, Thomas; Barbas, Carlos F

    2014-08-19

    The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein-DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular

  3. Associations between transcriptional changes and protein phenotypes provide insights into immune regulation in corals.

    Science.gov (United States)

    Fuess, Lauren E; Pinzόn C, Jorge H; Weil, Ernesto; Mydlarz, Laura D

    2016-09-01

    Disease outbreaks in marine ecosystems have driven worldwide declines of numerous taxa, including corals. Some corals, such as Orbicella faveolata, are particularly susceptible to disease. To explore the mechanisms contributing to susceptibility, colonies of O. faveolata were exposed to immune challenge with lipopolysaccharides. RNA sequencing and protein activity assays were used to characterize the response of corals to immune challenge. Differential expression analyses identified 17 immune-related transcripts that varied in expression post-immune challenge. Network analyses revealed several groups of transcripts correlated to immune protein activity. Several transcripts, which were annotated as positive regulators of immunity were included in these groups, and some were downregulated following immune challenge. Correlations between expression of these transcripts and protein activity results further supported the role of these transcripts in positive regulation of immunity. The observed pattern of gene expression and protein activity may elucidate the processes contributing to the disease susceptibility of species like O. faveolata. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  5. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    Science.gov (United States)

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-04

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  6. Protein regulation of induced pluripotent stem cells by transplanting in a Huntington's animal model.

    Science.gov (United States)

    Mu, S; Han, L; Zhou, G; Mo, C; Duan, J; He, Z; Wang, Z; Ren, L; Zhang, J

    2016-10-01

    The purpose of this study was to determine the functional recovery and protein regulation by transplanted induced pluripotent stem cells in a rat model of Huntington's disease (HD). In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle 10 days after the quinolinic acid injection. At 8 weeks after transplantation, fluorodeoxyglucose-PET/CT scan and balance-beam test were performed to evaluate the functional recovery of experimental rats. In addition, immunofluorescence and protein array analysis were used to investigate the regulation of stimulated protein expression in the striatum. At 8 weeks after induced pluripotent stem cell transplantation, motor function was improved in comparison with the quinolinic acid-treated rats. High fluorodeoxyglucose accumulation in the injured striatum was also observed by PET/CT scans. In addition, immunofluorescence analysis demonstrated that implanted cells migrated from the lateral ventricle into the lesioned striatum and differentiated into striatal projection neurons. Array analysis showed a significant upregulation of GFR (Glial cell line-derived neurotrophic factor receptor) alpha-1, Adiponectin/Acrp30, basic-fibroblast growth factors, MIP-1 (Macrophage-inflammatory protein) alpha and leptin, as well as downregulation of cytokine-induced neutrophil chemoattractant-3 in striatum after transplantatation of induced pluripotent stem cells in comparison with the quinolinic acid -treated rats. The findings in this work indicate that transplantation of induced pluripotent stem cells is a promising therapeutic candidate for HD. © 2016 British Neuropathological Society.

  7. Coordinate regulation of the mother centriole component nlp by nek2 and plk1 protein kinases.

    Science.gov (United States)

    Rapley, Joseph; Baxter, Joanne E; Blot, Joelle; Wattam, Samantha L; Casenghi, Martina; Meraldi, Patrick; Nigg, Erich A; Fry, Andrew M

    2005-02-01

    Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds gamma-tubulin, is a G(2)/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with gamma-tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G(2)/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.

  8. Nutritional regulation of muscle protein synthesis with resistance exercise: strategies to enhance anabolism

    Directory of Open Access Journals (Sweden)

    Churchward-Venne Tyler A

    2012-05-01

    Full Text Available Abstract Provision of dietary amino acids increases skeletal muscle protein synthesis (MPS, an effect that is enhanced by prior resistance exercise. As a fundamentally necessary process in the enhancement of muscle mass, strategies to enhance rates of MPS would be beneficial in the development of interventions aimed at increasing skeletal muscle mass particularly when combined with chronic resistance exercise. The purpose of this review article is to provide an update on current findings regarding the nutritional regulation of MPS and highlight nutrition based strategies that may serve to maximize skeletal muscle protein anabolism with resistance exercise. Such factors include timing of protein intake, dietary protein type, the role of leucine as a key anabolic amino acid, and the impact of other macronutrients (i.e. carbohydrate on the regulation of MPS after resistance exercise. We contend that nutritional strategies that serve to maximally stimulate MPS may be useful in the development of nutrition and exercise based interventions aimed at enhancing skeletal muscle mass which may be of interest to elderly populations and to athletes.

  9. [myc-p62 fusion protein suppresses the phosphorylation of extracellular signal-regulated kinase].

    Science.gov (United States)

    Zhang, Yunjing; Feng, Yingying; Zhang, Li; Xu, Xiaojie; Wang, Tao; Fan, Zhongyi; Ye, Qinong; Zhang, Rong

    2015-05-01

    To construct a eukaryotic expression vector of human autophagy maker protein P62 labeled with myc tag, and detect its biological function. Human P62 gene was amplified from human breast DNA library by PCR and cloned into pXJ-40-myc vector. HEK293T cells were transfected with the recombinant plasmid myc-p62. Western blotting was conducted to detect the fusion protein expression and the effect on the phosphorylation of extracellular signal-regulated kinase (ERK). Double enzyme identification and sequencing result showed that P62 eukaryotic expression vector labeled with myc tag was successfully constructed and the inserted fragment was correct. Western blotting indicated that the fusion protein was successfully expressed and was able to inhibit ERK phosphorylation. The eukaryotic expression vector of myc-p62 was successfully constructed and proved to have an inhibitory effect on ERK phosphorylation.

  10. Decoding sORF translation - from small proteins to gene regulation.

    Science.gov (United States)

    Cabrera-Quio, Luis Enrique; Herberg, Sarah; Pauli, Andrea

    2016-11-01

    Translation is best known as the fundamental mechanism by which the ribosome converts a sequence of nucleotides into a string of amino acids. Extensive research over many years has elucidated the key principles of translation, and the majority of translated regions were thought to be known. The recent discovery of wide-spread translation outside of annotated protein-coding open reading frames (ORFs) came therefore as a surprise, raising the intriguing possibility that these newly discovered translated regions might have unrecognized protein-coding or gene-regulatory functions. Here, we highlight recent findings that provide evidence that some of these newly discovered translated short ORFs (sORFs) encode functional, previously missed small proteins, while others have regulatory roles. Based on known examples we will also speculate about putative additional roles and the potentially much wider impact that these translated regions might have on cellular homeostasis and gene regulation.

  11. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  12. Abiotic stress responses in plants: roles of calmodulin-regulated proteins

    Science.gov (United States)

    Virdi, Amardeep S.; Singh, Supreet; Singh, Prabhjeet

    2015-01-01

    Intracellular changes in calcium ions (Ca2+) in response to different biotic and abiotic stimuli are detected by various sensor proteins in the plant cell. Calmodulin (CaM) is one of the most extensively studied Ca2+-sensing proteins and has been shown to be involved in transduction of Ca2+ signals. After interacting with Ca2+, CaM undergoes conformational change and influences the activities of a diverse range of CaM-binding proteins. A number of CaM-binding proteins have also been implicated in stress responses in plants, highlighting the central role played by CaM in adaptation to adverse environmental conditions. Stress adaptation in plants is a highly complex and multigenic response. Identification and characterization of CaM-modulated proteins in relation to different abiotic stresses could, therefore, prove to be essential for a deeper understanding of the molecular mechanisms involved in abiotic stress tolerance in plants. Various studies have revealed involvement of CaM in regulation of metal ions uptake, generation of reactive oxygen species and modulation of transcription factors such as CAMTA3, GTL1, and WRKY39. Activities of several kinases and phosphatases have also been shown to be modulated by CaM, thus providing further versatility to stress-associated signal transduction pathways. The results obtained from contemporary studies are consistent with the proposed role of CaM as an integrator of different stress signaling pathways, which allows plants to maintain homeostasis between different cellular processes. In this review, we have attempted to present the current state of understanding of the role of CaM in modulating different stress-regulated proteins and its implications in augmenting abiotic stress tolerance in plants. PMID:26528296

  13. Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans.

    Science.gov (United States)

    Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V; Nechipurenko, Inna V; Blacque, Oliver E; Sengupta, Piali

    2013-04-01

    The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.

  14. Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation

    Directory of Open Access Journals (Sweden)

    Lahiri Ansuman

    2011-09-01

    Full Text Available Abstract Background HIP1 Protein Interactor (HIPPI is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS, present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator. Results We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD. Conclusions Taken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a

  15. Interactions of SR45, an SR-like protein, with spliceosomal proteins and an intronic sequence: insights into regulated splicing.

    Science.gov (United States)

    Day, Irene S; Golovkin, Maxim; Palusa, Saiprasad G; Link, Alicia; Ali, Gul S; Thomas, Julie; Richardson, Dale N; Reddy, Anireddy S N

    2012-09-01

    SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  16. Localization and androgen regulation of metastasis-associated protein 1 in mouse epididymis.

    Directory of Open Access Journals (Sweden)

    Li Ma

    Full Text Available BACKGROUND: Metastasis-associated protein 1 (MTA1, the founding member of the MTA family of genes, can modulate transcription by influencing the status of chromatin remodeling. Despite its strong correlation with the metastatic potential of cancer cells, MTA1 can also regulate crucial cellular pathways by modifying the acetylation status. We have previously reported the presence of MTA1/MTA1 in human and mouse testes, providing the evidence for its involvement in the regulation of testicular function during murine spermatogenesis. The objective of present study was to further assess the localization of MTA1 in mouse epididymis on both transcriptional and translational level, and then to explore whether MTA1 expression is regulated by androgens and postnatal epididymal development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were deprived of circulating androgen by bilaterally castration and were then supplemented with exogenous testosterone propionate for one week. MTA1 was immunolocalized in the epithelium of the entire epididymis with the maximal expression in the nuclei of principal cells and of clear cells in proximal region. Its expression decreased gradually after castration, whereas testosterone treatment could restore the expression, indicating that the expression of this gene is dependent on androgen. During postnatal development, the protein expression in the epididymis began to appear from day 7 to day 14, increased dramatically from postnatal day 28, and peaked at adulthood onwards, coinciding with both the well differentiated status of epididymis and the mature levels of circulating androgens. This region- and cell-specific pattern was also conservative in normal human epididymis. CONCLUSIONS: Our data suggest that the expression of MTA1 protein could be regulated by androgen pathway and its expression level is closely associated with the postnatal development of the epididymis, giving rise to the possibility that this gene plays a

  17. Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [University of British Columbia, Vancouver; Wang, Shucai [University of British Columbia, Vancouver; Valerius, Oliver [Universitaet Goettingen, Goettingen, Germany; Hall, Hardy [University of British Columbia, Vancouver; Zeng, Qingning [University of British Columbia, Vancouver; Li, Jian-Feng [Harvard University; Weston, David [ORNL; Ellis, Brian [University of British Columbia, Vancouver; Chen, Jay [ORNL

    2011-01-01

    Earlier studies have shown that RACK1 functions as a negative regulator of ABA responses in Arabidopsis, but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, co-expression analysis revealed that >80% of the genes co-expressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1 s function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and yeast RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B and RACK1C, complemented the growth defects of the S. cerevisiae cpc2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor 6 (eIF6), whose mammalian homologue is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

  18. Myostatin inhibits eEF2K-eEF2 by regulating AMPK to suppress protein synthesis.

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    Deng, Zhao; Luo, Pei; Lai, Wen; Song, Tongxing; Peng, Jian; Wei, Hong-Kui

    2017-12-09

    Growth of skeletal muscle is dependent on the protein synthesis, and the rate of protein synthesis is mainly regulated in the stage of translation initiation and elongation. Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, is a negative regulator of protein synthesis. C2C12 myotubes was incubated with 0, 0.01, 0.1, 1, 2, 3 μg/mL myostatin recombinant protein, and then we detected the rates of protein synthesis by the method of SUnSET. We found that high concentrations of myostatin (2 and 3 μg/mL) inhibited protein synthesis by blocking mTOR and eEF2K-eEF2 pathway, while low concentration of myostatin (0.01, 0.1 and 1 μg/mL) regulated eEF2K-eEF2 pathway activity to block protein synthesis without affected mTOR pathway, and myostatin inhibited eEF2K-eEF2 pathway through regulating AMPK pathway to suppress protein synthesis. It provided a new mechanism for myostatin regulating protein synthesis and treating muscle atrophy. Copyright © 2017. Published by Elsevier Inc.

  19. Identification of novel mitosis regulators through data mining with human centromere/kinetochore proteins as group queries

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    Tipton Aaron R

    2012-06-01

    Full Text Available Abstract Background Proteins functioning in the same biological pathway tend to be transcriptionally co-regulated or form protein-protein interactions (PPI. Multiple spatially and temporally regulated events are coordinated during mitosis to achieve faithful chromosome segregation. The molecular players participating in mitosis regulation are still being unravelled experimentally or using in silico methods. Results An extensive literature review has led to a compilation of 196 human centromere/kinetochore proteins, all with experimental evidence supporting the subcellular localization. Sixty-four were designated as “core” centromere/kinetochore components based on peak expression and/or well-characterized functions during mitosis. By interrogating and integrating online resources, we have mined for genes/proteins that display transcriptional co-expression or PPI with the core centromere/kinetochore components. Top-ranked hubs in either co-expression or PPI network are not only enriched with known mitosis regulators, but also contain candidates whose mitotic functions are not yet established. Experimental validation found that KIAA1377 is a novel centrosomal protein that also associates with microtubules and midbody; while TRIP13 is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31comet. Conclusions Transcriptional co-expression and PPI network analyses with known human centromere/kinetochore proteins as a query group help identify novel potential mitosis regulators.

  20. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells.

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    Ming-Chih Lai

    Full Text Available Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia.

  1. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

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    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  2. DJ-1 protein regulates CD3+ T cell migration via overexpression of CXCR4 receptor.

    Science.gov (United States)

    Jung, Seung Hyo; Won, Kyung Jong; Lee, Kang Pa; Lee, Dong Hyun; Yu, Suyeol; Lee, Dong-Youb; Seo, Eun-Hye; Kang, Hyun; Park, Eun-Seok; Kim, Hyun-Joong; Lee, Seung Hyun; Kim, Bokyung

    2014-08-01

    DJ-1-a multifunctional protein responding to oxidative stress-is a possible regulator of the inflammatory response that plays an important role in atherosclerosis. Stromal cell-derived factor (SDF)-1 and its receptor, chemokine receptor type 4 (CXCR4), have been implicated in the recruitment of inflammatory cells during atherosclerosis. Here we investigated the hypothesis that DJ-1 protein might participate in CD3+ T cell functions in response to SDF-1 and contribute to the pathogenesis of atherosclerosis. SDF-1 stimulated migration in mouse CD3+ T cells in a dose-dependent manner. SDF-1 also elevated the phosphorylation level of extracellular-regulated kinase (ERK) 1/2 in CD3+ T cells. These SDF-1-induced responses were greater in CD3+ T cells from DJ-1 gene knockout (DJ-1(-/-)) mice than in those from wild type (DJ-1(+/+)) mice and were abolished by treatment with WZ811 and PD98059, inhibitors of CXCR4 and ERK1/2, respectively. Flow cytometry revealed that expression of the CXCR4 receptor was greater in CD3+ T cells from DJ-1(-/-) mice than in those from the controls. Moreover, expression of the CD3 protein was observed in the neointimal plaque from carotid artery-ligated mice and was stronger in DJ-1(-/-) mice compared with controls. The CD3+ T cell subsets, Th1 and Th17, showed increased production of interferon-γ and interleukin-17 in DJ-1(-/-) compared with DJ-1(+/+) mice. DJ-1 protein is involved in the SDF-1-induced CD3+ T cell migration via overexpression of the CXCR4 receptor, and that DJ-1 acts as an inhibitory regulator in vascular remodeling such as neointima formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

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    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  4. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

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    Yang, Hua Qian; Jana, Kundan; Rindler, Michael J; Coetzee, William A

    2017-11-13

    ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression

  5. The broad spectrum of signaling pathways regulated by unfolded protein response in neuronal homeostasis.

    Science.gov (United States)

    Saito, Atsushi; Imaizumi, Kazunori

    2017-06-28

    The protein folding capabilities in the endoplasmic reticulum (ER) are disturbed by alternations in the cellular homeostasis such as the disruption of calcium ion homeostasis, the expression of mutated proteins and oxidative stress. In response to these ER dysfunctions, eukaryotic cells activate canonical branches of signal transduction cascades to restore the protein folding capacity and avoid irreversible damages, collectively termed the unfolded protein response (UPR). Prolonged ER dysfunctions and the downregulation of UPR signaling pathways have been accepted as a crucial trigger for the pathogenesis of various neurodegenerative diseases. Furthermore, recent studies have revealed that the UPR has a wide spectrum of signaling pathways for unique physiological roles in the diverse developmental, differential and lipidomic processes. A developed and intricate ER network exists in the neurites of neurons. Neuronal ER functions and ER-derived signaling mediate efficient communication between cell soma and distal sites through local protein synthesis, sorting and lipogenesis. However, relevant of ER-derived UPR signaling pathways in the elaborate mechanisms regulating neuronal activities, synaptic functions and protective responses against injury is not fully elucidated. In this review, we summarized our current understanding of how the UPR functions provide the appropriate signals for neuronal capabilities. We also reviewed how UPR dysfunctions lead to the pathogenesis of neurodegenerative diseases, and the possibilities ameliorating their toxic effects by targeting UPR components. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

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    Rosner Marsha

    2007-01-01

    Full Text Available Abstract Raf Kinase Inhibitory Protein (RKIP is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.

  7. Characterisation of the salmon cystic fibrosis transmembrane conductance regulator protein for structural studies

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    Naomi L. Pollock

    2014-11-01

    Full Text Available The cystic fibrosis transmembrane conductance regulator protein (CFTR is a chloride channel highly expressed in the gills of Salmo salar, with a role in osmoregulation. It shares 60% identity with the human CFTR channel, mutations to which can cause the common genetic disorder cystic fibrosis CF. The expression and localisation of salmon CFTR have been investigated, but the isolated protein has not been extensively characterised. Here we present a protocol for the purification of recombinant salmon CFTR, along with biophysical and structural characterisation of the purified protein. Salmon CFTR was overexpressed in Saccharomyces cerevisiae, solubilised in the detergent LPG-14 and chromatographically purified by nickel-affinity and size-exclusion chromatography methods. Prior to size-exclusion chromatography samples of salmon CFTR had low purity, and contained large quantities of aggregated protein. Compared to size-exclusion chromatography profiles of other orthologues of CFTR, which had less evidence of aggregation, salmon CFTR appeared to have lower intrinsic stability than human and platypus CFTR. Nonetheless, repeated size-exclusion chromatography allowed monodisperse salmon CFTR to be isolated, and multi-angle light scattering was used to determine its oligomeric state. The monodispersity of the sample and its oligomeric state were confirmed using cryo-electron microscopy and small-angle X-ray scattering (SAXS. These data were also processed to calculate a low-resolution structure of the salmon CFTR, which showed similar architecture to other ATP-binding cassette proteins.

  8. Glucose-regulated protein 78 regulates the expression of mitochondrial genesis proteins in HBV-related hepatocellular carcinoma: a clinical analysis

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    LI Yaping

    2017-10-01

    Full Text Available ObjectiveTo investigate the expression of glucose-regulated protein 78 (GRP78 in HBV-related hepatocellular carcinoma (HBV-HCC and its association with clinicopathological features, as well as its regulatory effect on mitochondrial genesis proteins in hepatoma cells, and to provide a basis for new strategies for the prevention and treatment of HCC. MethodsTissue samples were collected from 54 patients with HBV-HCC, and immunohistochemistry and Western blot were used to measure the expression of GRP78, Lon, TFAM, and cytochrome C oxidase Ⅳ (COXⅣ. The expression of GRP78 in hepatoma cells was interfered by siRNA, and then the expression of GRP78, Lon, mitochondrial transcription factor A (TFAM, and COX Ⅳ was measured. Quantitative real-time PCR was used to measure the level of mitochondrial DNA (mtDNA in clinical specimens and HCC cells after GRP78 expression was interfered with. A statistical analysis was performed for clinical and experimental data. The t-test was used for comparison of continuous data between groups, the Fisher′s exact test was used for comparison of categorical data between groups, and the Kaplan-Meier method was used for survival analysis. Results Compared with the adjacent tissues, HBV-HCC tissues had significantly higher expression of GRP78 and Lon (t=9.135 and 5523, both P<0.0001 and significantly lower expression of the mitochondrial genesis proteins TFAM and COX Ⅳ and mtDNA level (t=2.765, 4260, and 12.280, P=0.011, <0.001, and <0.001. There were significant increases in the expression of the mitochondrial genesis proteins TFAM and COX Ⅳ and mtDNA level after the interference with GRP78 expression in hepatoma cells (all P<0.05. There were significant differences in the expression of GRP78 between patients with different numbers of tumors, patients with and without portal vein tumor thrombus, and patients with different tumor stages (P=0.016, 0.003, and 0.045. The patients with low GRP78

  9. Characterization of the heterotrimeric G-protein family and its transmembrane regulator from capsicum (Capsicum annuum L.).

    Science.gov (United States)

    Romero-Castillo, Rafael A; Roy Choudhury, Swarup; León-Félix, Josefina; Pandey, Sona

    2015-05-01

    Throughout evolution, organisms have created numerous mechanisms to sense and respond to their environment. One such highly conserved mechanism involves regulation by heterotrimeric G-protein complex comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits. In plants, these proteins play important roles in signal transduction pathways related to growth and development including response to biotic and abiotic stresses and consequently affect yield. In this work, we have identified and characterized the complete heterotrimeric G-protein repertoire in the Capsicum annuum (Capsicum) genome which consists of one Gα, one Gβ and three Gγ genes. We have also identified one RGS gene in the Capsicum genome that acts as a regulator of the G-protein signaling. Biochemical activities of the proteins were confirmed by assessing the GTP-binding and GTPase activity of the recombinant Gα protein and its regulation by the GTPase acceleration activity of the RGS protein. Interaction between different subunits was established using yeast- and plant-based analyses. Gene and protein expression profiles of specific G-protein components revealed interesting spatial and temporal regulation patterns, especially during root development and during fruit development and maturation. This research thus details the characterization of the first heterotrimeric G-protein family from a domesticated, commercially important vegetable crop. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

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    Douglas, Lois M; Wang, Hong X; Konopka, James B

    2013-11-26

    Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane

  11. Reciprocal regulation of protein synthesis and carbon metabolism for thylakoid membrane biogenesis.

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    Alexandra-Viola Bohne

    Full Text Available Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2, a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC, which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.

  12. Glucose-regulated protein 58 modulates β-catenin protein stability in a cervical adenocarcinoma cell line.

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    Liao, Chia-Jung; Wu, Tzu-I; Huang, Ya-Hui; Chang, Ting-Chang; Lai, Chyong-Huey; Jung, Shih-Ming; Hsueh, Chuen; Lin, Kwang-Huei

    2014-08-01

    Cervical cancer continues to threaten women's health worldwide, and the incidence of cervical adenocarcinoma (AD) is rising in the developed countries. Previously, we showed that glucose-regulated protein 58 (Grp58) served as an independent factor predictive of poor prognosis of patients with cervical AD. However, the molecular mechanism underlying the involvement of Grp58 in cervical carcinogenesis is currently unknown. DNA microarray and enrichment analysis were used to identify the pathways disrupted by knockdown of Grp58 expression. Among the pathway identified, the WNT signaling pathway was one of those that were significantly associated with knockdown of Grp58 expression in HeLa cells. Our experiments showed that β-catenin, a critical effector of WNT signaling, was stabilized thereby accumulated in stable Grp58 knockdown cells. Membrane localization of β-catenin was observed in Grp58 knockdown, but not control cells. Using a transwell assay, we found that accumulated β-catenin induced by Grp58 knockdown or lithium chloride treatment inhibited the migration ability of HeLa cells. Furthermore, an inverse expression pattern of Grp58 and β-catenin was observed in cervical tissues. Our results demonstrate that β-catenin stability is negatively regulated by Grp58 in HeLa cells. Overexpression of Grp58 may be responsible for the loss of or decrease in membranous β-catenin expression in cervical AD.

  13. Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration.

    Science.gov (United States)

    Ferreira-da-Silva, André; Valacca, Cristina; Rios, Elisabete; Pópulo, Helena; Soares, Paula; Sobrinho-Simões, Manuel; Scorrano, Luca; Máximo, Valdemar; Campello, Silvia

    2015-01-01

    Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1--a papillary thyroid carcinoma derived cell line--and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells' migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype.

  14. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

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    Maia M Chan

    Full Text Available Hematopoietic protein-1 (Hem-1 is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein complex, which regulates filamentous actin (F-actin polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A, which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  15. Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

    Science.gov (United States)

    Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

  16. A multifunctional protein EWS regulates the expression of Drosha and microRNAs

    Science.gov (United States)

    Kim, K Y; Hwang, Y J; Jung, M-K; Choe, J; Kim, Y; Kim, S; Lee, C-J; Ahn, H; Lee, J; Kowall, N W; Kim, Y K; Kim, J-I; Lee, S B; Ryu, H

    2014-01-01

    EWS (Ewing's Sarcoma) gene encodes an RNA/DNA-binding protein that is ubiquitously expressed and involved in various cellular processes. EWS deficiency leads to impaired development and early senescence through unknown mechanisms. We found that EWS regulates the expression of Drosha and microRNAs (miRNAs). EWS deficiency resulted in increased expression of Drosha, a well-known microprocessor, and increased levels of miR-29b and miR-18b. Importantly, miR-29b and miR-18b were directly involved in the post-transcriptional regulation of collagen IV alpha 1 (Col4a1) and connective tissue growth factor (CTGF) in EWS knock-out (KO) mouse embryonic fibroblast cells. The upregulation of Drosha, miR-29b and miR-18b and the sequential downregulation of Col4a1 and CTGF contributed to the deregulation of dermal development in EWS KO mice. Otherwise, knockdown of Drosha rescued miRNA-dependent downregulation of Col4a1 and CTGF proteins. Taken together, our data indicate that EWS is involved in post-transcriptional regulation of Col4a1 and CTGF via a Drosha–miRNA-dependent pathway. This finding suggests that EWS has a novel role in dermal morphogenesis through the modulation of miRNA biogenesis. PMID:24185621

  17. The polarity protein Scribble regulates myelination and remyelination in the central nervous system.

    Directory of Open Access Journals (Sweden)

    Andrew A Jarjour

    2015-03-01

    Full Text Available The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS, requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK/mitogen-activated protein kinase (MAP kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination.

  18. Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

    Energy Technology Data Exchange (ETDEWEB)

    M Teplova; J Song; H Gaw; A Teplov; D Patel

    2011-12-31

    CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

  19. Down-regulation of cardiac lineage protein (CLP-1) expression in CLP-1 +/- mice affords.

    Science.gov (United States)

    Mascareno, Eduardo; Manukyan, Irena; Das, Dipak K; Siddiqui, M A Q

    2009-08-01

    In order to understand the transcriptional mechanism that underlies cell protection to stress, we evaluated the role of CLP-1, a known inhibitor of the transcription elongation complex (pTEFb), in CLP-1 +/- mice hearts. Using the isolated heart model, we observed that the CLP-1 +/- hearts, when subjected to ischaemic stress and evaluated by haemodynamic measurements, exhibit significant cardioprotection. CLP-1 remains associated with the pTEFb complex in the heterozygous hearts, where as it is released in the wild-type hearts suggesting the involvement of pTEFb regulation in cell protection. There was a decrease in Cdk7 and Cdk9 kinase activity and consequently in phosphorylation of serine-5 and serine-2 of Pol II CTD in CLP-1 +/- hearts. However, the levels of mitochondrial proteins, PGC-1alpha and HIF-1alpha, which enhance mitochondrial activity and are implicated in cell survival, were increased in CLP-1 +/- hearts subjected to ischaemic stress compared to that in wild-type CLP-1 +/- hearts treated identically. There was also an increase in the expression of pyruvate dehydrogenase kinase (PDK-1), which facilitates cell adaptation to hypoxic stress. Taken together, our data suggest that regulation of the CLP-1 levels is critical to cellular adaptation of the survival program that protects cardiomyocytes against stress due collectively to a decrease in RNA Pol II phosphorylation but an increase in expression of target proteins that regulate mitochondrial function and metabolic adaptation to stress.

  20. Inter-regulation of the unfolded protein response and auxin signaling.

    Science.gov (United States)

    Chen, Yani; Aung, Kyaw; Rolčík, Jakub; Walicki, Kathryn; Friml, Jiří; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is a signaling network triggered by overload of protein-folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down-regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species-specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER-localized auxin transporters, including PIN5, we define a long-neglected biological significance of ER-based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone-dependent strategy for coordinating ER function with physiological processes. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  1. Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

    Science.gov (United States)

    Zhao, Fangzhou; Yu, Chien-Hung; Liu, Yi

    2017-08-21

    Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

    Science.gov (United States)

    Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

    2011-11-01

    Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

  3. Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani

    DEFF Research Database (Denmark)

    Jensen, A T; Curtis, J; Montgomery, J

    2001-01-01

    To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identif......To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family...

  4. 24-hour oscillation of mouse methionine aminopeptidase2, a regulator of tumor progression, is regulated by clock gene proteins.

    Science.gov (United States)

    Nakagawa, Hiroo; Koyanagi, Satoru; Takiguchi, Takako; Kuramoto, Yukako; Soeda, Shinji; Shimeno, Hiroshi; Higuchi, Shun; Ohdo, Shigehiro

    2004-11-15

    Methionine aminopeptidase2 (MetAP2) plays an important role in the growth of endothelial cells during the tumor angiogenesis stage. Recently, we have clarified that mouse methionine aminopeptidases (mMetAPs) show a 24-hour rhythm in implanted tumor masses. In the present study, we investigated the mechanism underlying the 24-hour rhythm of mMetAP2 activity in tumor-bearing mice under a light-dark (lights on from 7 a.m. to 7 p.m.) cycle. The 5' flanking region of mMetAP2 included eight E-boxes. The transcription of the mMetAP2 promoter was enhanced by the mCLOCK:mBMAL1 heterodimer, and its activation was inhibited by mPER2 or mCRY1. Deletion and mutation of the E-boxes in the region indicated that the E-box nearest to the initiation start site played an important role in the transcriptional regulation by clock genes. In sarcoma180-bearing mice, the pattern of binding of mCLOCK and mBMAL1 to the E-box and transcription of the mMetAP2 promoter showed a 24-hour rhythm with higher levels from the mid-light to early dark phase. The pattern of mMetAP2 transcription was closely associated with that of mMetAP2 mRNA expression in three types of tumor-bearing mice. mMetAP2 protein expression varied with higher levels from the late-dark to early light phase. The rhythmicity of the protein expression was synchronous with that of the activity of mMetAPs but out of phase with that of the mMetAP2 mRNA expression. These results suggest that the 24-hour rhythm of mMetAP2 activity is regulated by the transcription of clock genes within the clock feedback loops.

  5. Compatibility of Astragalus and Salvia extract inhibits myocardial fibrosis and ventricular remodeling by regulation of protein kinase D1 protein

    Science.gov (United States)

    Mao, Bingyu; Nuan, Liu; Yang, Lei; Zeng, Xiaotao

    2015-01-01

    Aims: This study is to determine the effect of astragalus and salvia extract on the alteration of myocardium in a rat model of myocardial infarction. Methods: A total of 40 male Sprague-Dawley rats were randomly divided into the sham-operated group, the control group, the Astragalus group, the Salvia group, and the compatibility of Astragalus and Salvia and group. The cardiac functions were determined at 8 weeks after treatment. Hematoxylin-eosin staining was performed to observe the morphology and arrangement of cardiomyocytes. Masson’s trichrome staining was performed to investigate the distribution of myocardial interstitial collagen. Immunohistochemical staining was performed to determine the expression ofprotein kinase D1 in myocardial tissues. Results: In the sham-operated group, the Astragalus group, the Salvia group, and the compatibility of Astragalus and Salvia group, the left ventricular systolic pressure and the maximum rate of left ventricular pressure were significantly increased while the left ventricular end diastolic pressure were significantly decreased when compared with those in the control group (P Salvia group. Contents of collagen fibers in myocardial tissues were decreased in the compatibility of Astragalus and Salvia group (P Salvia group. Conclusions: Compatibility of Astragalus and Salvia extract may inhibit myocardial fibrosis and ventricular remodeling by regulation of protein kinase D1 protein in a rat model of myocardial infarction. PMID:26064267

  6. Bone morphogenetic protein-7 expression is down-regulated in human clear cell renal carcinoma.

    Science.gov (United States)

    Basic-Jukic, Nikolina; Hudolin, Tvrtko; Radic-Antolic, Margareta; Coric, Marijana; Zadro, Renata; Kastelan, Zeljko; Pasini, Josip; Bandic-Pavlovic, Daniela; Kes, Petar

    2011-01-01

    Recent studies demonstrated that the expression pattern of bone morphogenetic protein-7 (BMP-7) is altered in different tumors. We determined expression of BMP-7 in human clear cell renal carcinoma (CCRC). Samples from cancer and corresponding healthy tissue were obtained from 20 patients who underwent nephrectomy for CCRC. Expression of BMP-7 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR), and protein expression was analyzed by immunohistochemistry. RT-PCR showed strong down-regulation of BMP-7 mRNA in cancer tissue. Immunohistochemistry revealed expression of BMP-7 in normal renal tissue, with almost complete loss of BMP-7 expression in malignant cells of 6 patients (30%). After 3 years of follow-up, 5 out of 6 patients with high BMP-7 mRNA expression were alive and disease-free, compared with 9 out of 14 patients with low BMP-7 mRNA expression. BMP-7 mRNA and protein expression were down-regulated in CCRC. Further prospective studies are needed to characterize the role of BMP-7 in human CCRC.

  7. Regulated transport into the nucleus of herpesviridae DNA replication core proteins.

    Science.gov (United States)

    Gualtiero, Alvisi; Jans, David A; Camozzi, Daria; Avanzi, Simone; Loregian, Arianna; Ripalti, Alessandro; Palù, Giorgio

    2013-09-16

    The Herpesvirdae family comprises several major human pathogens belonging to three distinct subfamilies. Their double stranded DNA genome is replicated in the nuclei of infected cells by a number of host and viral products. Among the latter the viral replication complex, whose activity is strictly required for viral replication, is composed of six different polypeptides, including a two-subunit DNA polymerase holoenzyme, a trimeric primase/helicase complex and a single stranded DNA binding protein. The study of herpesviral DNA replication machinery is extremely important, both because it provides an excellent model to understand processes related to eukaryotic DNA replication and it has important implications for the development of highly needed antiviral agents. Even though all known herpesviruses utilize very similar mechanisms for amplification of their genomes, the nuclear import of the replication complex components appears to be a heterogeneous and highly regulated process to ensure the correct spatiotemporal localization of each protein. The nuclear transport process of these enzymes is controlled by three mechanisms, typifying the main processes through which protein nuclear import is generally regulated in eukaryotic cells. These include cargo post-translational modification-based recognition by the intracellular transporters, piggy-back events allowing coordinated nuclear import of multimeric holoenzymes, and chaperone-assisted nuclear import of specific subunits. In this review we summarize these mechanisms and discuss potential implications for the development of antiviral compounds aimed at inhibiting the Herpesvirus life cycle by targeting nuclear import of the Herpesvirus DNA replicating enzymes.

  8. Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis.

    Science.gov (United States)

    Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E; Seidman, J G; Pu, William T; Wang, Da-Zhi

    2015-11-02

    Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

  9. Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2

    Directory of Open Access Journals (Sweden)

    Yaoming Yang

    2012-06-01

    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 feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2 in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1. USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.

  10. Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2.

    Science.gov (United States)

    Yang, Yaoming; Duguay, David; Bédard, Nathalie; Rachalski, Adeline; Baquiran, Gerardo; Na, Chan Hyun; Fahrenkrug, Jan; Storch, Kai-Florian; Peng, Junmin; Wing, Simon S; Cermakian, Nicolas

    2012-08-15

    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 feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1). USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.

  11. Cysteine-rich protein 1 (CRP1 regulates actin filament bundling

    Directory of Open Access Journals (Sweden)

    Fraley Tamara S

    2005-12-01

    Full Text Available Abstract Background Cysteine-rich protein 1 (CRP1 is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. Results CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. Conclusion CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.

  12. Protein phosphatase 1 regulates the histone code for long-term memory.

    Science.gov (United States)

    Koshibu, Kyoko; Gräff, Johannes; Beullens, Monique; Heitz, Fabrice D; Berchtold, Dominik; Russig, Holger; Farinelli, Mélissa; Bollen, Mathieu; Mansuy, Isabelle M

    2009-10-14

    Chromatin remodeling through histone posttranslational modifications (PTMs) and DNA methylation has recently been implicated in cognitive functions, but the mechanisms involved in such epigenetic regulation remain poorly understood. Here, we show that protein phosphatase 1 (PP1) is a critical regulator of chromatin remodeling in the mammalian brain that controls histone PTMs and gene transcription associated with long-term memory. Our data show that PP1 is present at the chromatin in brain cells and interacts with enzymes of the epigenetic machinery including HDAC1 (histone deacetylase 1) and histone demethylase JMJD2A (jumonji domain-containing protein 2A). The selective inhibition of the nuclear pool of PP1 in forebrain neurons in transgenic mice is shown to induce several histone PTMs that include not only phosphorylation but also acetylation and methylation. These PTMs are residue-specific and occur at the promoter of genes important for memory formation like CREB (cAMP response element-binding protein) and NF-kappaB (nuclear factor-kappaB). These histone PTMs further co-occur with selective binding of RNA polymerase II and altered gene transcription, and are associated with improved long-term memory for objects and space. Together, these findings reveal a novel mechanism for the epigenetic control of gene transcription and long-term memory in the adult brain that depends on PP1.

  13. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

    Directory of Open Access Journals (Sweden)

    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

  14. TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs.

    Science.gov (United States)

    Kim, Hyeung; Lee, Ok-Hee; Xin, Huawei; Chen, Liuh-Yow; Qin, Jun; Chae, Heekyung Kate; Lin, Shiaw-Yih; Safari, Amin; Liu, Dan; Songyang, Zhou

    2009-04-01

    In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain-containing telomeric proteins TRF1 and TRF2 associate with a collection of molecules necessary for telomere maintenance and cell-cycle progression. However, the specificity and the mechanisms by which TRF2 communicates with different signaling pathways remain largely unknown. Using oriented peptide libraries, we demonstrate that the TRFH domain of human TRF2 recognizes [Y/F]XL peptides with the consensus motif YYHKYRLSPL. Disrupting the interactions between the TRF2 TRFH domain and its targets resulted in telomeric DNA-damage responses. Furthermore, our genome-wide target analysis revealed phosphatase nuclear targeting subunit (PNUTS) and microcephalin 1 (MCPH1) as previously unreported telomere-associated proteins that directly interact with TRF2 via the [Y/F]XL motif. PNUTS and MCPH1 can regulate telomere length and the telomeric DNA-damage response, respectively. Our findings indicate that an array of TRF2 molecules functions as a protein hub and regulates telomeres by recruiting different signaling molecules via a linear sequence code.

  15. Phosphorylation and Ubiquitination Regulate Protein Phosphatase 5 Activity and Its Prosurvival Role in Kidney Cancer

    Directory of Open Access Journals (Sweden)

    Natela Dushukyan

    2017-11-01

    Full Text Available The serine/threonine protein phosphatase 5 (PP5 regulates multiple cellular signaling networks. A number of cellular factors, including heat shock protein 90 (Hsp90, promote the activation of PP5. However, it is unclear whether post-translational modifications also influence PP5 phosphatase activity. Here, we show an “on/off switch” mechanism for PP5 regulation. The casein kinase 1δ (CK1δ phosphorylates T362 in the catalytic domain of PP5, which activates and enhances phosphatase activity independent of Hsp90. Overexpression of the phosphomimetic T362E-PP5 mutant hyper-dephosphorylates substrates such as the co-chaperone Cdc37 and glucocorticoid receptor in cells. Our proteomic approach revealed that the tumor suppressor von Hippel-Lindau protein (VHL interacts with and ubiquitinates K185/K199-PP5 for proteasomal degradation in a hypoxia- and prolyl-hydroxylation-independent manner. Finally, VHL-deficient clear cell renal cell carcinoma (ccRCC cell lines and patient tumors exhibit elevated PP5 levels. Downregulation of PP5 causes ccRCC cells to undergo apoptosis, suggesting a prosurvival role for PP5 in kidney cancer.

  16. The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway.

    Directory of Open Access Journals (Sweden)

    John S Bett

    Full Text Available Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA. AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.

  17. Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    O'Ryan, Liam; Rimington, Tracy; Cant, Natasha; Ford, Robert C

    2012-03-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein(1-3). Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed (4-9). Proteins involved in the processing of recombinant CFTR in yeast have been described(6-9) .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)(10). The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for

  18. Rab35 protein regulates evoked exocytosis of endothelial Weibel–Palade bodies

    Science.gov (United States)

    Biesemann, Anja; Gorontzi, Alexandra; Barr, Francis; Gerke, Volker

    2017-01-01

    Weibel–Palade bodies (WPB) are secretory organelles of endothelial cells that undergo evoked exocytosis following intracellular Ca2+ or cAMP elevation, thereby supplying the vasculature with factors controlling hemostasis. Several cytosolic and membrane-associated proteins, including the Rab family members Rab3, Rab15, and Rab27a, have been implicated in regulating the acute exocytosis of WPB. Here, we carried out a genome-wide screen to identify Rab pathways affecting WPB exocytosis. Overexpression of a specific subset of Rab GTPase–activating proteins (RabGAPs) inhibited histamine-evoked, Ca2+-dependent WPB exocytosis, presumably by inactivating the target Rab GTPases. Among these RabGAPs, we concentrated on TBC1D10A and showed that the inhibitory effect depends on its GAP activity. We confirmed that Rab35 was a target Rab of TBC1D10A in human endothelial cells; Rab35 interacted with TBC1D10A, and expression of the GAP-insensitive Rab35(Q67A) mutant rescued the inhibitory effect of TBC1D10A overexpression on WPB exocytosis. Furthermore, knockdown of Rab35 and expression of a dominant-negative Rab35 mutant both inhibited histamine-evoked secretion of the WPB cargos von Willebrand factor and P-selectin. Pulldown and co-immunoprecipitation experiments identified the ArfGAP with coiled-coil, Ank repeat, and pleckstrin homology domain–containing protein ACAP2 as an Rab35 effector in endothelial cells, and depletion as well as overexpression approaches revealed that ACAP2 acts as a negative regulator of WPB exocytosis. Interestingly, a known ACAP2 target, the small GTPase Arf6, supported histamine-evoked WPB exocytosis, as shown by knockdown and overexpression of a dominant-negative Arf6 mutant. Our data identify Rab35 as a novel regulator of WPB exocytosis, most likely acting through the downstream effectors ACAP2 and Arf6. PMID:28566286

  19. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

    Science.gov (United States)

    Yang, Su; Xu, Hong; Wang, Jiali; Liu, Chengzhi; Lu, Huizhi; Liu, Mengjia; Zhao, Ye; Tian, Bing; Wang, Liangyan; Hua, Yuejin

    2016-01-01

    The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998), Lon proteases (dr0349 and dr1974), NADH-quinone oxidoreductase (dr1506), thiosulfate sulfurtransferase (dr2531), the DNA repair protein UvsE (dr1819), PprA (dra0346), and RecN (dr1447), are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  20. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Su Yang

    Full Text Available The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2 treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998, Lon proteases (dr0349 and dr1974, NADH-quinone oxidoreductase (dr1506, thiosulfate sulfurtransferase (dr2531, the DNA repair protein UvsE (dr1819, PprA (dra0346, and RecN (dr1447, are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  1. SUMOylation of GTF2IRD1 regulates protein partner interactions and ubiquitin-mediated degradation.

    Directory of Open Access Journals (Sweden)

    Jocelyn Widagdo

    Full Text Available GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxβ, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxβ. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxβ itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell.

  2. The biophysical and biochemical properties of the autoimmune regulator (AIRE) protein.

    Science.gov (United States)

    Perniola, Roberto; Musco, Giovanna

    2014-02-01

    AIRE (for autoimmune regulator) is a multidomain protein that performs a fundamental function in the thymus and possibly in the secondary lymphoid organs: the regulation, especially in the sense of activation, of the process of gene transcription in cell lines deputed to the presentation of self-antigens to the maturing T lymphocytes. The apoptosis of the elements bearing T-cell receptors with critical affinity for the exhibited self-antigens prevents the escape of autoreactive clones and represents a simple and efficient mechanism of deletional self-tolerance. However, AIRE action relies on an articulated complex of biophysical and biochemical properties, in most cases attributable to single subspecialized domains. Here a thorough review of the matter is presented, with a privileged look at the pathogenic changes of AIRE that interfere with such properties and lead to the impairment in its chief function. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

    Science.gov (United States)

    Gil-Torregrosa, B; Urdiales, J L; Lozano, J; Mates, J M; Sanchez-Jimenez, F

    1994-08-08

    Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.

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

    DEFF Research Database (Denmark)

    Gallina, Irene

    and Cmr1 in yeast Saccharomyces cerevisiae, two novel players in the maintenance of genome integrity, which act through modulation of turnover or stability of repair and replication proteins. In S. cerevisiae, RAD56 belongs to the RAD52 epistasis group of genetic loci that confer X-ray but not UV......Mechanisms of surveillance of DNA replication and DNA repair are essential for genome stability and cell viability. Both the integrity of the repair pathways and the timely regulation of the repair mechanisms are essential for the maintenance of genome stability. One of the layers of regulation...... 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...

  5. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...

  6. Hypothalamic AMP-activated Protein Kinase as a Regulator of Food Intake and Energy Balance.

    Science.gov (United States)

    Oh, Tae Seok; Jeon, Yoonjeong; Kim, Seolsong; Kim, Eun-Kyoung

    2016-01-01

    The maintenance of appetite at proper levels, depending on the energy status, is important; otherwise abnormal appetite may cause a series of disorders, such as anorexia, hyperphagia, obesity, and its complications (diabetes mellitus, hypertension, cardiovascular disease, and fatty liver disease). Hypothalamic AMPactivated protein kinase (AMPK) integrates diverse hormonal and nutritional signals to regulate food intake and energy metabolism. Recent evidence suggests that different hormones, nutrients and synthetic chemicals can modulate AMPK activity in the hypothalamus, thereby regulating food intake and body weight, through neuropeptide expressions. In order to elucidate the mechanisms that control hypothalamic AMPK activity, a variety of studies have focused on finding upstream and downstream modulators of hypothalamic AMPK for the regulation of food intake and energy balance. This review highlights the current evidence for understanding how hypothalamic AMPK regulates food intake and energy balance, and will help in the development of effective interventions for the treatment of food intake-related disorders. In the future, it is hoped that new pharmaceutical developments targeting hypothalamic AMPK, in combination with careful clinical trials, will lead to improved and effective therapeutic strategies for complications caused by abnormal appetite and energy balance.

  7. Cross-regulation of protein stability by p53 and nuclear receptor SHP.

    Directory of Open Access Journals (Sweden)

    Zhihong Yang

    Full Text Available We report here a novel interplay between tumor suppressor p53 and nuclear receptor SHP that controls p53 and SHP stability. Overexpression of p53 causes rapid SHP protein degradation, which does not require the presence of Mdm2 and is mediated by the proteosome pathway. Overexpressing SHP alone does not affect p53 stability. However, SHP destabilizes p53 by augmentation of Mdm2 ubiquitin ligase activity toward p53. The single amino acid substitution in the SHP protein SHPK170R increases SHP binding to p53 relative to SHP wild-type, whereas SHPG171A variant shows a diminished p53 binding. As a result of the cross-regulation, the tumor suppressor function of p53 and SHP in inhibition of colon cancer growth is compromised. Our findings reveal a unique scenario for a cross-inhibition between two tumor suppressors to keep their expression and function in check.

  8. Complementary RNA and Protein Profiling Identifies Iron as a Key Regulator of Mitochondrial Biogenesis

    Directory of Open Access Journals (Sweden)

    Jarred W. Rensvold

    2013-01-01

    Full Text Available Mitochondria are centers of metabolism and signaling whose content and function must adapt to changing cellular environments. The biological signals that initiate mitochondrial restructuring and the cellular processes that drive this adaptive response are largely obscure. To better define these systems, we performed matched quantitative genomic and proteomic analyses of mouse muscle cells as they performed mitochondrial biogenesis. We find that proteins involved in cellular iron homeostasis are highly coordinated with this process and that depletion of cellular iron results in a rapid, dose-dependent decrease of select mitochondrial protein levels and oxidative capacity. We further show that this process is universal across a broad range of cell types and fully reversed when iron is reintroduced. Collectively, our work reveals that cellular iron is a key regulator of mitochondrial biogenesis, and provides quantitative data sets that can be leveraged to explore posttranscriptional and posttranslational processes that are essential for mitochondrial adaptation.

  9. The PERK arm of the unfolded protein response regulates satellite cell-mediated skeletal muscle regeneration.

    Science.gov (United States)

    Xiong, Guangyan; Hindi, Sajedah M; Mann, Aman K; Gallot, Yann S; Bohnert, Kyle R; Cavener, Douglas R; Whittemore, Scott R; Kumar, Ashok

    2017-03-23

    Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

  10. Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation

    Science.gov (United States)

    Vogelmann, Jutta; Valeri, Alessandro; Guillou, Emmanuelle; Cuvier, Olivier; Nollmann, Marcelo

    2013-01-01

    Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization. PMID:21983085

  11. Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation.

    Science.gov (United States)

    Runfola, Valeria; Sebastian, Soji; Dilworth, F Jeffrey; Gabellini, Davide

    2015-02-15

    Among the Mef2 family of transcription factors, Mef2D is unique in that it undergoes tissue-specific splicing to generate an isoform that is essential for muscle differentiation. However, the mechanisms mediating this muscle-specific processing of Mef2D remain unknown. Using bioinformatics, we identified Rbfox proteins as putative modulators of Mef2D muscle-specific splicing. Accordingly, we found direct and specific Rbfox1 and Rbfox2 binding to Mef2D pre-mRNA in vivo. Gain- and loss-of-function experiments demonstrated that Rbfox1 and Rbfox2 cooperate in promoting Mef2D splicing and subsequent myogenesis. Thus, our findings reveal a new role for Rbfox proteins in regulating myogenesis through activation of essential muscle-specific splicing events. © 2015. Published by The Company of Biologists Ltd.

  12. 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...... of the circadian clock, both at the level of the core pacemaker and its response to external cues....... feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free...

  13. Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus▿

    OpenAIRE

    Black, Wesley P.; Schubot, Florian D.; Li, Zhuo; Yang, Zhaomin

    2010-01-01

    Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS re...

  14. MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

    Science.gov (United States)

    Rodriguez Peña, Marcelo J.; Castillo Bennett, Jimena V.; Soler, Osvaldo M.; Mayorga, Luis S.; Michaut, Marcela A.

    2013-01-01

    Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering

  15. Hamster oviductin regulates tyrosine phosphorylation of sperm proteins during in vitro capacitation.

    Science.gov (United States)

    Saccary, Laurelle; She, Yi-Min; Oko, Richard; Kan, Frederick W K

    2013-08-01

    Oviductin or OVGP1, also known as oviduct-specific glycoprotein, has been shown to enhance sperm capacitation in addition to its other beneficial effects on fertilization and early embryo development. We hypothesized that estrus stage-specific hamster oviductin (eHamOVGP1) can potentiate the enhancement of tyrosine phosphorylation of sperm proteins during capacitation. Immunofluorescent staining and confocal microscopy as well as immunocytochemistry and surface replica technique localized tyrosine-phosphorylated proteins to the equatorial segment and midpiece after incubation of hamster sperm in capacitation medium in the presence or absence of eHamOVGP1. Increase of tyrosine phosphorylation level in the equatorial segment occurred as early as 5 min after incubation in the presence of eHamOVGP1. Immunostaining for eHamOVGP1 further increased upon prolonged incubation of sperm in medium containing the glycoprotein. Regardless of the presence or absence of eHamOVGP1, phosphotyrosine expression was observed along the tail, particularly at the midpiece. Western blotting of NP40-extracted sperm proteins (25, 37, and 44 kDa) and NP40-non-extractable sperm proteins (70, 83, 90 kDa) showed increased immunolabeling intensity after 5, 60, 120, and 180 min of capacitation in the presence of eHamOVGP1. Mass spectrometric analysis identified several proteins of functions known to be involved in metabolic pathways responsible for enhancement of tyrosine phosphorylation in its presence. The present investigation provides evidence that eHamOVGP1 regulates the expression of protein tyrosine phosphorylation in sperm capacitated in vitro, further supporting an important role of the presence of OVGP1 in the oviductal milieu during the process of fertilization.

  16. Bumble bees regulate their intake of essential protein and lipid pollen macronutrients.

    Science.gov (United States)

    Vaudo, A D; Stabler, D; Patch, H M; Tooker, J F; Grozinger, C M; Wright, G A

    <