DOCSLIB.ORG

Regulation and Function of the RPEL Protein – Phactr1 Maria Katarzyna

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation and Function of the RPEL Protein – Phactr1 Maria Katarzyna Regulation and function of the RPEL protein – Phactr1 Maria Katarzyna Wiezlak University College London and Cancer Research UK London Research Institute PhD Supervisor: Richard Treisman A thesis submitted for the degree of Doctor of Philosophy University College London May 2013 Declaration I Maria Wiezlak confirm that the work presented in this thesis is my own. Part of the work was performed in collaboration. In Chapter 3, experiments presented in Figure 3.11 and parts of Figures 3.8, 3.10 and 3.15 were performed by Jessica Diring, a postdoc from the Transcription Laboratory at the London Research Institute. The structural analysis presented in Chapter 4 was performed in collaboration with Stephane Mouilleron, a postdoc from Neil McDonald’s Laboratory (Structural Biology Laboratory at the London Research Institute). Of the work presented, I was involved in initial optimisation of complex formation and initial screens. Stephane Mouilleron performed majority of the crystallisation screens and the optimisation work at the crystallisation stage, collected the X-ray diffraction data and solved the crystal structures. He also performed SEC- MALLS analysis of the complexes. Where analyses were performed by Stephane Mouilleron, this is indicated in the figure legends. In Chapter 5, experiments presented in parts of Figures 5.3, 5.4, 5.5 and 5.6 were performed by Jasmine Abella, a postdoc from Michael Way’s Laboratory (Cell Motility Laboratory at the London Research Institute). All other experiments presented in this thesis were performed by the author. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Results presented in this thesis have been published in: Wiezlak M, Diring J, Abella J, Mouilleron S, Way M, McDonald NQ, Treisman R. (2012). G-actin regulates shuttling and PP1 binding by the RPEL protein Phactr1 to control actomyosin assembly. Journal of Cell Science 125, 5860-5872. Mouilleron S*, Wiezlak M*, O'Reilly N, Treisman R, McDonald NQ. (2012). Structures of the Phactr1 RPEL domain and RPEL motif complexes with G-actin reveal the molecular basis for actin binding cooperativity. Structure 20, 1960-70. *equal contribution 2 Abstract Actin-binding proteins play well established roles in the regulation of actin dynamics and assembly of F-actin based structures involved in cell motility and adhesion. The Phosphatase and actin regulator (Phactr) family of proteins each contain four G-actin binding RPEL motifs and has been found to bind protein phosphatase 1 (PP1) via their C-terminal domain. Their function is not well established and it has been unclear whether G-actin can be their regulator. Members of the Phactr family are highly expressed in the nervous system and in some metastatic cancers. The RPEL domain was previously shown to confer Rho-regulated nuclear shuttling and activation of Serum Response Factor (SRF) coactivator myocardin – related transcription factor A (MRTF-A, also known as MAL/MKL1). MRTF-A is cytoplasmic in unstimulated cells and accumulates in the nucleus upon activation of Rho-actin signalling. In this thesis I show that activation of Rho-actin signalling by serum stimulation induces nuclear accumulation of Phactr1, but not other Phactr family members (Phactr2-4). Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is Importin α−β-dependent. I also reveal that G-actin and Importin α−β bind competitively to nuclear import signals associated with the N- and C-terminal RPEL motifs in Phactr1. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation by elevated G-actin. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and expression of Phactr1 C-terminal RPEL mutants that cannot bind G-actin induces actomyosin foci dependent on PP1 binding. In CHL-1 metastatic melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility, and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability. 3 Acknowledgement I would like to thank Richard Treisman for giving me the opportunity to work in his laboratory, for his supervision, support, and advice. This work would never have been possible without the help of my collaborators. Many thanks to Stephane Mouilleron, who has been an excellent colleague and collaborator, always creative and helpful. I would also like to thank Jessica Diring, who offered her help with biochemical and mutational studies presented in Chapter 3. I am very grateful to Jasmine Abella, who helped with functional studies. Her experience and imaging skills greatly improved the work presented in Chapter 5. I thank my thesis committee and collaborators, Neil McDonald and Michael Way, who offered their continuous support, help, and creativity during this project. Work presented here would not be possible without the continuous help and advice of everyone in the lab. I thank Cyril for fruitful discussions, Diane and Patrick for great advice and comic relief, and Rob and Matthew for day-to-day help with almost everything! I also thank Ricky, Francesco, Anastasia, Charlie and past members of the lab. Special thanks go to Rafal, who helped me in the lab, when I started. Many thanks to Lucy, who has been extremely helpful with all the admin issues during my time at the LRI. Thanks to Nicola O’Reilly for her expertise in peptide synthesis and to everyone in the LRI equipment park. Many thanks to Sally and Sophie (and Sabina and Erin), the graduate team at the LRI, for making this experience so enjoyable. I thank Cancer Research UK for funding. Most importantly, would like to thank my family and friends. I would never have been able to do this work without my mom and my parents in law, who helped every day to take care of my little daughter Mia and were enormously supportive. I would also like to thank my grandpa, who encouraged me to pursue science. Finally, I would like to thank my husband Jas, who has been extremely patient throughout my studies. 4 Table of Contents Abstract ................................................................................................................ 3 Acknowledgement .............................................................................................. 4 Table of Contents ................................................................................................ 5 Table of figures ................................................................................................. 10 List of tables ...................................................................................................... 13 Abbreviations .................................................................................................... 14 Chapter 1. Introduction .................................................................................. 19 1.1 Functions of actin ............................................................................................... 19 1.1.1 The dynamics of actin turnover .................................................................... 19 1.1.2 Actin control by Rho-GTPases ..................................................................... 24 1.1.3 Actin binding proteins and their roles ........................................................... 33 1.1.4 Actin-binding proteins involved in actin filament nucleation and dynamics.37 1.1.5 Actomyosin contractility ............................................................................... 44 1.2 Actin in transcription regulation – regulation of SRF cofactors MRTFs ..... 55 1.2.1 SRF transcription factor ................................................................................ 56 1.2.2 MRTF family of SRF cofactors .................................................................... 62 1.2.3 Protein interaction and domain organisation of MRTFs ............................... 64 1.2.4 Actin-dependent regulation of MRTF localisation and activity ................... 68 1.2.5 The RPEL motif defines a G-actin binding element. .................................... 72 1.3 Protein Phosphatase 1 (PP1) ............................................................................. 79 1.3.1 The structure of PP1 catalytic subunit .......................................................... 80 1.3.2 Substrate specificity, recognition and inhibition mechanisms ...................... 83 1.3.3 PP1 holoenzymes .......................................................................................... 85 1.3.4 Regulatory binding sites ................................................................................ 86 1.3.5 Functions of PP1 regulatory subunits ........................................................... 90 1.4 The Phactr family of G-actin binding PP1 cofactors ...................................... 98 1.4.1 Early studies of Phactr family ....................................................................... 98 1.4.2 Phactr family domain organisation and reported interactions ..................... 100 1.4.3 Expression and tissue distribution ............................................................... 107 1.4.4 Subcellular localisation ............................................................................... 109 5 1.4.5 Proposed functions of Phactr proteins .......................................................
Load more

Recommended publications
  • Deficiency of Macrophage PHACTR1 Impairs Efferocytosis and Promotes Atherosclerotic Plaque Necrosis
    Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis Canan Kasikara, … , Muredach P Reilly, Ira Tabas J Clin Invest. 2021. https://doi.org/10.1172/JCI145275. Research In-Press Preview Cardiology Cell biology Graphical abstract Find the latest version: https://jci.me/145275/pdf Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis Canan Kasikara,1 Maaike Schilperoort,1 Brennan Gerlach,1 Chenyi Xue,1 Xiaobo Wang1, Ze Zheng,2 George Kuriakose,1 Bernhard Dorweiler,3 Hanrui Zhang,1 Gabrielle Fredman4, Danish Saleheen,1 Muredach P. Reilly1,5, and Ira Tabas1,6,7 1Department of Medicine, Columbia University Irving Medical Center, New York, New York, USA. 2Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. 3Department of Vascular Surgery, University of Cologne, Cologne, Germany.4Deparment of Molecular and Cellular Physiology, Albany Medical Center, Albany, New York, USA. 5Irving Institute for Clinical and Translational Research, Columbia University Irving Medical Center, New York, New York, USA. 6Department of Physiology and Cellular Biophysics and 7Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, New York, USA. Address correspondence to: Ira Tabas, Depart of Medicine, Columbia University Irving Medical Center, 630 W. 168th Street, New York, New York 10032, USA. Phone: 212.305.9430; E-mail: [email protected]. 1 Abstract Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains a common variant rs9349379 (A > G) associated with CAD.
    [Show full text]
  • PHACTR1 Genetic Variability Is Not Critical in Small Vessel Ischemic
    www.nature.com/scientificreports OPEN PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice Clemens Messerschmidt2, Marco Foddis1, Sonja Blumenau1, Susanne Müller1, Kajetan Bentele2, Manuel Holtgrewe2, Celia Kun‑Rodrigues3, Isabel Alonso4, Maria do Carmo Macario5, Ana Sofa Morgadinho5, Ana Graça Velon6, Gustavo Santo5,8, Isabel Santana5,7,8, Saana Mönkäre9,10, Liina Kuuluvainen9,11, Johanna Schleutker10, Minna Pöyhönen9,11, Liisa Myllykangas12, Assunta Senatore13, Daniel Berchtold1, Katarzyna Winek1, Andreas Meisel1, Aleksandra Pavlovic14, Vladimir Kostic14, Valerija Dobricic14,15, Ebba Lohmann16,17,18, Hasmet Hanagasi16, Gamze Guven19, Basar Bilgic16, Jose Bras3, Rita Guerreiro3, Dieter Beule2, Ulrich Dirnagl1 & Celeste Sassi1,20* Recently, several genome‑wide association studies identifed PHACTR1 as key locus for fve diverse vascular disorders: coronary artery disease, migraine, fbromuscular dysplasia, cervical artery dissection and hypertension. Although these represent signifcant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi‑ethnic cohort of 180 early‑onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL‑like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by diferent degrees of PcomAs 1Department of Experimental Neurology, Center for Stroke Research Berlin (CSB), Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Charitéplatz 1, 10117 Berlin, Germany.
    [Show full text]
  • Regulators at Every Step—How Micrornas Drive Tumor Cell Invasiveness and Metastasis
    cancers Review Regulators at Every Step—How microRNAs Drive Tumor Cell Invasiveness and Metastasis 1,2,3, 1,2, 1, Tomasz M. Grzywa y , Klaudia Klicka y and Paweł K. Włodarski * 1 Department of Methodology, Medical University of Warsaw, 02-091 Warsaw, Poland; [email protected] (T.M.G.); [email protected] (K.K.) 2 Doctoral School, Medical University of Warsaw, 02-091 Warsaw, Poland 3 Department of Immunology, Medical University of Warsaw, 02-097 Warsaw, Poland * Correspondence: [email protected] These authors contributed equally to this work. y Received: 19 November 2020; Accepted: 7 December 2020; Published: 10 December 2020 Simple Summary: Tumor cell invasiveness and metastasis are key processes in cancer progression and are composed of many steps. All of them are regulated by multiple microRNAs that either promote or suppress tumor progression. Multiple studies demonstrated that microRNAs target the mRNAs of multiple genes involved in the regulation of cell motility, local invasion, and metastatic niche formation. Thus, microRNAs are promising biomarkers and therapeutic targets in oncology. Abstract: Tumor cell invasiveness and metastasis are the main causes of mortality in cancer. Tumor progression is composed of many steps, including primary tumor growth, local invasion, intravasation, survival in the circulation, pre-metastatic niche formation, and metastasis. All these steps are strictly controlled by microRNAs (miRNAs), small non-coding RNA that regulate gene expression at the post-transcriptional level. miRNAs can act as oncomiRs that promote tumor cell invasion and metastasis or as tumor suppressor miRNAs that inhibit tumor progression. These miRNAs regulate the actin cytoskeleton, the expression of extracellular matrix (ECM) receptors including integrins and ECM-remodeling enzymes comprising matrix metalloproteinases (MMPs), and regulate epithelial–mesenchymal transition (EMT), hence modulating cell migration and invasiveness.
    [Show full text]
  • Camp Receptor Protein–Camp Plays a Crucial Role in Glucose–Lactose Diauxie by Activating the Major Glucose Transporter Gene in Escherichia Coli
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12914–12919, November 1997 Biochemistry cAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in Escherichia coli KEIKO KIMATA*, HIDEYUKI TAKAHASHI*, TOSHIFUMI INADA*, PIETER POSTMA†, AND HIROJI AIBA*‡ *Department of Molecular Biology, School of Science, Nagoya University, Chikusa, Nagoya 464-01, Japan; and †E. C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands Communicated by Sydney Kustu, University of California, Berkeley, CA, September 17, 1997 (received for review May 2, 1997) ABSTRACT The inhibition of b-galactosidase expression certain conditions, for example, when added to cells growing in a medium containing both glucose and lactose is a typical on a poor carbon source such as glycerol or succinate (6, 7). example of the glucose effect in Escherichia coli. We studied the Glucose is thought to reduce cAMP level by decreasing the glucose effect in the lacL8UV5 promoter mutant, which is phosphorylated form of enzyme IIAGlc, which is proposed to independent of cAMP and cAMP receptor protein (CRP). A be involved in the activation of adenylate cyclase (3–5). strong inhibition of b-galactosidase expression by glucose and Glucose also is known to reduce the CRP level through the a diauxic growth were observed when the lacL8UV5 cells were autoregulation of the crp gene (7–10). grown on a glucose–lactose medium. The addition of isopropyl When E. coli finds both glucose and lactose in the medium, b-D-thiogalactoside to the culture medium eliminated the it preferentially uses the glucose, and the use of lactose is glucose effect.
    [Show full text]
  • Deficiency of Macrophage PHACTR1 Impairs Efferocytosis and Promotes Atherosclerotic Plaque Necrosis
    Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis Canan Kasikara, … , Muredach P. Reilly, Ira Tabas J Clin Invest. 2021;131(8):e145275. https://doi.org/10.1172/JCI145275. Research Article Cardiology Cell biology Graphical abstract Find the latest version: https://jci.me/145275/pdf The Journal of Clinical Investigation RESEARCH ARTICLE Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis Canan Kasikara,1 Maaike Schilperoort,1 Brennan Gerlach,1 Chenyi Xue,1 Xiaobo Wang,1 Ze Zheng,2 George Kuriakose,1 Bernhard Dorweiler,3 Hanrui Zhang,1 Gabrielle Fredman,4 Danish Saleheen,1 Muredach P. Reilly,1,5 and Ira Tabas1,6,7 1Department of Medicine, Columbia University Irving Medical Center, New York, New York, USA. 2Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. 3Department of Vascular Surgery, University of Cologne, Cologne, Germany. 4Department of Molecular and Cellular Physiology, Albany Medical Center, Albany, New York, USA. 5Irving Institute for Clinical and Translational Research, Columbia University Irving Medical Center, New York, New York, USA. 6Department of Physiology and Cellular Biophysics and 7Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, New York, USA. Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin- binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis.
    [Show full text]
  • I = Chpt 15. Positive and Negative Transcriptional Control at Lac BMB
    BMB 400 Part Four - I = Chpt 15. Positive and Negative Transcriptional Control at lac B M B 400 Part Four: Gene Regulation Section I = Chapter 15 POSITIVE AND NEGATIVE CONTROL SHOWN BY THE lac OPERON OF E. COLI A. Definitions and general comments 1. Operons An operon is a cluster of coordinately regulated genes. It includes structural genes (generally encoding enzymes), regulatory genes (encoding, e.g. activators or repressors) and regulatory sites (such as promoters and operators). 2. Negative versus positive control a. The type of control is defined by the response of the operon when no regulatory protein is present. b. In the case of negative control, the genes in the operon are expressed unless they are switched off by a repressor protein. Thus the operon will be turned on constitutively (the genes will be expressed) when the repressor in inactivated. c. In the case of positive control, the genes are expressed only when an active regulator protein, e.g. an activator, is present. Thus the operon will be turned off when the positive regulatory protein is absent or inactivated. Table 4.1.1. Positive vs. negative control BMB 400 Part Four - I = Chpt 15. Positive and Negative Transcriptional Control at lac 3. Catabolic versus biosynthetic operons a. Catabolic pathways catalyze the breakdown of nutrients (the substrate for the pathway) to generate energy, or more precisely ATP, the energy currency of the cell. In the absence of the substrate, there is no reason for the catabolic enzymes to be present, and the operon encoding them is repressed. In the presence of the substrate, when the enzymes are needed, the operon is induced or de-repressed.
    [Show full text]
  • Discovery and Systematic Characterization of Risk Variants and Genes For
    medRxiv preprint doi: https://doi.org/10.1101/2021.05.24.21257377; this version posted June 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 Discovery and systematic characterization of risk variants and genes for 2 coronary artery disease in over a million participants 3 4 Krishna G Aragam1,2,3,4*, Tao Jiang5*, Anuj Goel6,7*, Stavroula Kanoni8*, Brooke N Wolford9*, 5 Elle M Weeks4, Minxian Wang3,4, George Hindy10, Wei Zhou4,11,12,9, Christopher Grace6,7, 6 Carolina Roselli3, Nicholas A Marston13, Frederick K Kamanu13, Ida Surakka14, Loreto Muñoz 7 Venegas15,16, Paul Sherliker17, Satoshi Koyama18, Kazuyoshi Ishigaki19, Bjørn O Åsvold20,21,22, 8 Michael R Brown23, Ben Brumpton20,21, Paul S de Vries23, Olga Giannakopoulou8, Panagiota 9 Giardoglou24, Daniel F Gudbjartsson25,26, Ulrich Güldener27, Syed M. Ijlal Haider15, Anna 10 Helgadottir25, Maysson Ibrahim28, Adnan Kastrati27,29, Thorsten Kessler27,29, Ling Li27, Lijiang 11 Ma30,31, Thomas Meitinger32,33,29, Sören Mucha15, Matthias Munz15, Federico Murgia28, Jonas B 12 Nielsen34,20, Markus M Nöthen35, Shichao Pang27, Tobias Reinberger15, Gudmar Thorleifsson25, 13 Moritz von Scheidt27,29, Jacob K Ulirsch4,11,36, EPIC-CVD Consortium, Biobank Japan, David O 14 Arnar25,37,38, Deepak S Atri39,3, Noël P Burtt4, Maria C Costanzo4, Jason Flannick40, Rajat M 15 Gupta39,3,4, Kaoru Ito18, Dong-Keun Jang4,
    [Show full text]
  • (CRP) on Porin Genes and Its Own Gene in Yersinia Pestis
    Gao et al. BMC Microbiology 2011, 11:40 http://www.biomedcentral.com/1471-2180/11/40 RESEARCHARTICLE Open Access Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis He Gao1†, Yiquan Zhang1†, Lin Yang1,2, Xia Liu1,2, Zhaobiao Guo1, Yafang Tan1, Yanping Han1, Xinxiang Huang2, Dongsheng Zhou1*, Ruifu Yang1* Abstract Background: The cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the ompR-envZ operon in E. coli directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on ompR-envZ. Auto-regulation of CRP has also been established in E. coli. However, the regulation of porin genes and its own gene by CRP remains unclear in Y. pestis. Results: Y. pestis employs a distinct mechanism indicating that CRP has no regulatory effect on the ompR-envZ operon; however, it stimulates ompC and ompF directly, while repressing ompX. No transcriptional regulatory association between CRP and its own gene can be detected in Y. pestis, which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in E. coli. It is likely that Y. pestis OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently. Conclusion: Although the CRP of Y. pestis shows a very high homology to that of E. coli, and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the Y.
    [Show full text]
  • Fluorescence Spectroscopic Analysis of Ppgpp Binding to Camp Receptor Protein and Histone-Like Nucleoid Structuring Protein
    International Journal of Molecular Sciences Communication Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein Taner Duysak 1 , Thanh Tuyen Tran 1 , Aqeel Rana Afzal 2 and Che-Hun Jung 1,2,3,* 1 Department of Molecular Medicine, Chonnam National University, Gwangju 61186, Korea; [email protected] (T.D.); [email protected] (T.T.T.) 2 Department of Medical Science, Chonnam National University, Gwangju 61186, Korea; [email protected] 3 Department of Chemistry, Chonnam National University, Gwangju 61186, Korea * Correspondence: [email protected]; Tel.: +82-62-530-3383 Abstract: The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site Citation: Duysak, T.; Tran, T.T.; was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory Afzal, A.R.; Jung, C.-H.
    [Show full text]
  • The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression
    The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression. Kelloniemi, Annina; Szabo, Zoltan; Serpi, Raisa; Näpänkangas, Juha; Ohukainen, Pauli; Tenhunen, Olli; Kaikkonen, Leena; Koivisto, Elina; Bagyura, Zsolt; Kerkelä, Risto; Leosdottir, Margrét; Hedner, Thomas; Melander, Olle; Ruskoaho, Heikki; Rysä, Jaana Published in: PLoS ONE DOI: 10.1371/journal.pone.0130502 2015 Link to publication Citation for published version (APA): Kelloniemi, A., Szabo, Z., Serpi, R., Näpänkangas, J., Ohukainen, P., Tenhunen, O., Kaikkonen, L., Koivisto, E., Bagyura, Z., Kerkelä, R., Leosdottir, M., Hedner, T., Melander, O., Ruskoaho, H., & Rysä, J. (2015). The Early- Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression. PLoS ONE, 10(6), [e0130502]. https://doi.org/10.1371/journal.pone.0130502 Total number of authors: 15 General rights Unless other specific re-use rights are stated the following general rights apply: Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Read more about Creative commons licenses: https://creativecommons.org/licenses/ Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
    [Show full text]
  • Associations Between PHACTR1 Gene Polymorphisms and Pulse Pressure in Chinese Han Population
    Bioscience Reports (2020) 40 BSR20193779 https://doi.org/10.1042/BSR20193779 Research Article Associations between PHACTR1 gene polymorphisms and pulse pressure in Chinese Han population Kunfang Gu, Yue Zhang, Ke Sun and Xiubo Jiang Department of Epidemiology and Health Statistics, the School of Public Health of Qingdao University, No. 38 Dengzhou Road, Qingdao 266021, China Correspondence: Xiubo Jiang ([email protected]) Downloaded from http://portlandpress.com/bioscirep/article-pdf/40/6/BSR20193779/883367/bsr-2019-3779.pdf by guest on 01 October 2021 A genome-wide association study (GWAS) in Chinese twins was performed to explore asso- ciations between genes and pulse pressure (PP) in 2012, and detected a suggestive asso- ciation in the phosphatase and actin regulator 1 (PHACTR1) gene on chromosome 6p24.1 (rs1223397, P=1.04e−07). The purpose of the present study was to investigate associations of PHACTR1 gene polymorphisms with PP in a Chinese population. We recruited 347 sub- jects with PP ≥ 65 mmHg as cases and 359 subjects with 30 ≤ PP ≤ 45 mmHg as controls. Seven single nucleotide polymorphisms (SNPs) in the PHACTR1 gene were genotyped. Lo- gistic regression was performed to explore associations between SNPs and PP in codom- inant, additive, dominant, recessive and overdominant models. The Pearson’s χ2 test was applied to assess the relationships of haplotypes and PP. The A allele of rs9349379 had a positive effect on high PP. Multivariate logistic regression analysis showed that rs9349379 was significantly related to high PP in codominant [AA vs GG, 2.255 (1.132–4.492)], additive [GG vs GA vs AA, 1.368 (1.049–1.783)] and recessive [AA vs GA + GG, 2.062 (1.051–4.045)] models.
    [Show full text]
  • Glycine Cleavage System and Camp Receptor Protein Co-Regulate CRISPR/Cas3 Expression to Resist Bacteriophage
    viruses Article Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage Denghui Yang 1, Zhaofei Wang 1, Jingjiao Ma 1 , Qiang Fu 1, Lifei Wu 1, Hengan Wang 1, Shaohui Wang 2, Yaxian Yan 1,* and Jianhe Sun 1,* 1 Shanghai Key Laboratory of Veterinary Biotechnology, Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] (D.Y.); [email protected] (Z.W.); [email protected] (J.M.); [email protected] (Q.F.); [email protected] (L.W.); [email protected] (H.W.) 2 Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China; [email protected] * Correspondence: [email protected] (Y.Y.); [email protected] (J.S.); Tel.: +86-21-3420-6003 (Y.Y.); +86-21-3420-6926 (J.S.) Received: 5 December 2019; Accepted: 9 January 2020; Published: 13 January 2020 Abstract: The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression.
    [Show full text]