DOCSLIB.ORG

Analysis of the Pumilio Repression Mechanism and Its Impact on the Transcriptome

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Analysis of the Pumilio Repression Mechanism and Its Impact on the Transcriptome Analysis of the Pumilio Repression Mechanism and its Impact on the Transcriptome A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Rebecca J. Haugen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor Dr. Aaron Goldstrohm Committee members: Dr. Anja Bielinsky, Dr. Eric A. Hendrickson, Dr. David Greenstein May 2021 Rebecca J. Haugen Copyright 2021 Acknowledgements I would like to begin by acknowledging my husband Travis, for his ceaseless and unwavering support on this long journey. None of this would have been possible without him. I also thank my kids, Cody, Sarah, and Grace, for sacrificing some of their “Mom time” to all-day writing sprees or late nights in the lab. To Cody, for showing a keen interest in the science and allowing me to practice explaining it to him; to Sarah, for being my angel of sanity with relentless encouragement; to Grace, for providing comedic relief, and also engaging in late-night science talks: you have all been more instrumental in this accomplishment than I could ever explain. I would also like to thank my parents for their support and encouragement. I would like to thank my advisor Dr. Aaron Goldstrohm, for his guidance, and seemingly bottomless reservoir of patience and flexibility. I also thank my committee members Drs. Anja Bielinsky, Eric A. Hendrickson, and David Greenstein, for their encouragement, help, and feedback. I would like to especially thank Dr. Greenstein for supporting me and believing in me. I want to also thank the members of the Goldstrohm lab, past and present for their insight, encouragement, and scientific discussion, especially Dr. Katie McKenney, and my bench-mate, Rob Connacher. i Dedication This work is dedicated to my daughters, Sarah and Grace, and my son Cody. May you find enjoyment in working hard for something. ii Abstract The expression of genes in an organism is controlled by many methods. One of the most important means of regulation is post-transcriptional control of messenger RNA (mRNA). RNA binding proteins (RBPs) are crucial for this process. Here we describe the characterization of a functional domain within the Drosophila RBP Pumilio (Pum) and examine the effects of Pum on the transcriptome. Pum proteins are a highly conserved family of RBPs that control the expression of mRNA by binding to specific motifs in the 3’-UTR of transcripts. Through associations with other proteins, Pum proteins regulate the fate of an RNA, primarily causing decay of the target. Drosophila Pum contains autonomous domains capable of enacting decay independently from its RNA binding domain. The most conserved domain is repression domain 3 (RD3) which we characterize here. Using conservation analysis, we define conserved regions within RD3, and show their importance for the repressive activity of RD3. We identify two specific amino acids, F1033 and F1040, which are necessary for RD3’s function. Using yeast two-hybrid assays, we show that RD3 contacts several members of the CNOT deadenylase complex, the central scaffold unit Not1, and subunits Not2 and Not3. In Drosophila cells, we show that RD3 can interact with the CNOT complex. We further explore the endogenous targets of Drosophila Pum proteins by performing RNA sequencing experiments in which we deplete Pum and members of the CNOT complex. This is the first global analysis of the functional regulation of Pum targets in Drosophila. From this analysis we find that thousands of genes rely upon the regulatory framework provided by Pum. We demonstrate specific regulation of several targets of Pum using reporter assays and identify functional Pum binding sites within target 3’-UTRs. Finally, we show that regulation by Pum also affects the levels of expressed protein for two important targets, Pde11 and Raf. The work shown here enhances our understanding of Pum proteins for all organisms and the field of post- transcriptional control. iii Table of Contents Acknowledgements ............................................................................................... i Dedication .............................................................................................................ii Abstract ................................................................................................................ iii Table of Contents .................................................................................................iv List of Tables ....................................................................................................... vii List of Figures ..................................................................................................... viii List of Abbreviations ............................................................................................ xii Chapter 1: Introduction ......................................................................................... 1 1.1. Making the mRNA ................................................................................................ 1 1.2. Characteristics of mRNA ...................................................................................... 5 1.3. Cytoplasmic fates of mRNA ................................................................................. 8 1.3.1. Translation ..................................................................................................... 8 1.3.2. Storage and localization ................................................................................ 9 1.3.3. Messenger RNA decay .................................................................................10 1.4. Pumilio and PUF proteins....................................................................................13 1.4.1. Essential roles of Pum proteins ....................................................................14 1.4.2. Regulation of Pum ........................................................................................17 1.4.2.1. Regulation of Pum RNA .........................................................................17 1.4.2.2. Regulation of Pum protein ......................................................................18 1.4.3. Protein interactors of Pum ............................................................................20 1.4.4. The structure of Pum proteins .......................................................................21 Chapter 2: Structure-function analysis of RD3.................................................... 24 2.1. Introduction .........................................................................................................24 2.2. Overall structure of RD3 ......................................................................................26 2.3. Identification of conserved residues in RD3 ........................................................26 2.4. Functional activity of conserved regions ..............................................................31 2.4.1. Truncations of RD3 .......................................................................................32 2.4.2. Deletions of conserved regions .....................................................................36 2.4.3. Alanine scanning of conserved regions ........................................................40 2.4.4. Sufficiency of CR1 and CR4 .........................................................................46 2.5. Summary and discussion ....................................................................................49 2.6. Materials and methods ........................................................................................52 Chapter 3: Interactions of RD3 with the CNOT complex .................................... 56 iv 3.1. Introduction .........................................................................................................56 3.2. Interaction screens - Y2H assays ........................................................................57 3.3. Immunoprecipitation assays in Drosophila cells ..................................................71 3.4. Summary and discussion ....................................................................................76 3.5. Materials and methods ........................................................................................78 3.5.1. CRISPR generation of Not1-Flag Dl1 cells ...................................................82 Chapter 4: Endogenous targets of Pum proteins ................................................ 86 4.1. Introduction .........................................................................................................86 4.2. Identification of endogenous Pum targets ...........................................................87 4.3. Validation of Pum targets ....................................................................................91 4.4. Contribution of RD3 to repression of endogenous targets ...................................92 4.5. Pum depletion causes an increase in target protein levels ..................................97 4.6. Not1 as the effector of Pum-mediated decay of endogenous targets ..................99 4.7. Summary and discussion .................................................................................. 101 4.7.1. Biological significance of targets ................................................................. 102 4.8. Materials and methods ...................................................................................... 106 4.8.1. Quantitative western blotting ......................................................................
Load more

Recommended publications
  • Dissertation Characterization Of
    DISSERTATION CHARACTERIZATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES IN THE TRANSCRIPTOME OF THE CRUSTACEAN MOLTING GLAND Submitted by Nada Mukhtar Rifai Department of Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Spring 2019 Doctoral Committee: Advisor: Donald L. Mykles Deborah Garrity Shane Kanatous Santiago Di-Pietro Copyright by Nada Mukhtar Rifai 2019 All Rights Reserved ABSTRACT CHARACTERIZATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES IN THE TRANSCRIPTOME OF THE CRUSTACEAN MOLTING GLAND Molting in crustaceans is a complex physiological process that has to occur in order for the animal to grow. The old exoskeleton must be discarded and a new one to be formed from the inside out. Molting is coordinated and regulated mainly by two hormones; steroid hormones named ecdysteroids, which are synthesized and secreted from a pair of Y- organs (YOs) that are located in the cephalothorax and a neuropeptide hormone, the molt inhibiting hormone (MIH), which is secreted from the X-organ/sinus gland complex located in the eyestalks. Molting is induced when MIH is decreased in the blood (hemolymph) which in turn stimulates the YOs to produce and secrete ecdysteroids (molting hormones). There are four distinctive physiological states that the YO can be in throughout the molt cycle; the transition of the YO from the “basal” to the “activated” state happens when the animal enters premolt. During mid-premolt, the YO transitions to the “committed” state, in which the YO becomes insensitive to MIH. In this state, the circulating hemolymph contains high levels of ecdysteroids, which increase to a peak before the actual molt (ecdysis) happens.
    [Show full text]
  • Chemical Genetic Studies of Chemical Modulators of Mammalian Adenylyl Cyclases and Phosphodiesterases Expressed in Fission Yeast
    Chemical Genetic Studies of Chemical Modulators of Mammalian Adenylyl Cyclases and Phosphodiesterases Expressed in Fission Yeast Author: Ana Santos de Medeiros Persistent link: http://hdl.handle.net/2345/bc-ir:106786 This work is posted on eScholarship@BC, Boston College University Libraries. Boston College Electronic Thesis or Dissertation, 2016 Copyright is held by the author, with all rights reserved, unless otherwise noted. Boston College Morrisey College of Arts and Sciences Graduate School Department of Biology CHEMICAL GENETIC STUDIES OF CHEMICAL MODULATORS OF MAMMALIAN ADENYLYL CYCLASES AND PHOSPHODIESTERASES EXPRESSED IN FISSION YEAST a dissertation by ANA SANTOS DE MEDEIROS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2016 © copyright by ANA SANTOS DE MEDEIROS 2016 ABSTRACT Cyclic adenosine monophosphate (cAMP) and the second messengers that modulate several biological processes are regulated by adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterases (PDEs). ACs and PDEs are comprised of superfamilies of enzymes that are viewed as druggable targets due to their many distinct biological roles and tissue-specific distribution. As such, small molecule regulators of ACs and PDEs are important as chemical probes to study the roles of individual ACs or PDEs and as potential therapeutics. In the past, our lab has expressed 15 mammalian PDE genes in S. pombe, replacing the endogenous Cgs2 PDE. High throughput screens for PDE inhibitors identified novel compounds that show relevant biological activity in mammalian cell culture assays. The aim of this thesis is to develop tools to understand the mechanism of interaction between key regulators of the cAMP pathway and small molecules.
    [Show full text]
  • Phosphodiesterase (PDE)
    Phosphodiesterase (PDE) Phosphodiesterase (PDE) is any enzyme that breaks a phosphodiester bond. Usually, people speaking of phosphodiesterase are referring to cyclic nucleotide phosphodiesterases, which have great clinical significance and are described below. However, there are many other families of phosphodiesterases, including phospholipases C and D, autotaxin, sphingomyelin phosphodiesterase, DNases, RNases, and restriction endonucleases, as well as numerous less-well-characterized small-molecule phosphodiesterases. The cyclic nucleotide phosphodiesterases comprise a group of enzymes that degrade the phosphodiester bond in the second messenger molecules cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains. PDEs are therefore important regulators ofsignal transduction mediated by these second messenger molecules. www.MedChemExpress.com 1 Phosphodiesterase (PDE) Inhibitors, Activators & Modulators (+)-Medioresinol Di-O-β-D-glucopyranoside (R)-(-)-Rolipram Cat. No.: HY-N8209 ((R)-Rolipram; (-)-Rolipram) Cat. No.: HY-16900A (+)-Medioresinol Di-O-β-D-glucopyranoside is a (R)-(-)-Rolipram is the R-enantiomer of Rolipram. lignan glucoside with strong inhibitory activity Rolipram is a selective inhibitor of of 3', 5'-cyclic monophosphate (cyclic AMP) phosphodiesterases PDE4 with IC50 of 3 nM, 130 nM phosphodiesterase. and 240 nM for PDE4A, PDE4B, and PDE4D, respectively. Purity: >98% Purity: 99.91% Clinical Data: No Development Reported Clinical Data: No Development Reported Size: 1 mg, 5 mg Size: 10 mM × 1 mL, 10 mg, 50 mg (R)-DNMDP (S)-(+)-Rolipram Cat. No.: HY-122751 ((+)-Rolipram; (S)-Rolipram) Cat. No.: HY-B0392 (R)-DNMDP is a potent and selective cancer cell (S)-(+)-Rolipram ((+)-Rolipram) is a cyclic cytotoxic agent. (R)-DNMDP, the R-form of DNMDP, AMP(cAMP)-specific phosphodiesterase (PDE) binds PDE3A directly.
    [Show full text]
  • Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients with Stable Coronary Heart Disease
    Supplementary Online Content Ganz P, Heidecker B, Hveem K, et al. Development and validation of a protein-based risk score for cardiovascular outcomes among patients with stable coronary heart disease. JAMA. doi: 10.1001/jama.2016.5951 eTable 1. List of 1130 Proteins Measured by Somalogic’s Modified Aptamer-Based Proteomic Assay eTable 2. Coefficients for Weibull Recalibration Model Applied to 9-Protein Model eFigure 1. Median Protein Levels in Derivation and Validation Cohort eTable 3. Coefficients for the Recalibration Model Applied to Refit Framingham eFigure 2. Calibration Plots for the Refit Framingham Model eTable 4. List of 200 Proteins Associated With the Risk of MI, Stroke, Heart Failure, and Death eFigure 3. Hazard Ratios of Lasso Selected Proteins for Primary End Point of MI, Stroke, Heart Failure, and Death eFigure 4. 9-Protein Prognostic Model Hazard Ratios Adjusted for Framingham Variables eFigure 5. 9-Protein Risk Scores by Event Type This supplementary material has been provided by the authors to give readers additional information about their work. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Supplemental Material Table of Contents 1 Study Design and Data Processing ......................................................................................................... 3 2 Table of 1130 Proteins Measured .......................................................................................................... 4 3 Variable Selection and Statistical Modeling ........................................................................................
    [Show full text]
  • 1 PDE5 Inhibition Improves Symptom-Free Survival and Restores
    bioRxiv preprint doi: https://doi.org/10.1101/248187; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. PDE5 inhibition improves symptom-free survival and restores transverse tubule loss and catecholamine responsiveness in heart failure 1 1 Michael Lawless , Jessica L. Caldwell , Emma J. Radcliffe, George W.P. Madders, David C. 2 2 Hutchings, Lori S. Woods, Stephanie J. Church , Richard D. Unwin , Graeme J. Kirkwood, Lorenz K. Becker, Charles M. Pearman, Rebecca F. Taylor, David A. Eisner, Katharine M. * Dibb & Andrew. W Trafford . 1. Contributed Equally * Correspondence 2. Unit of Cardiac Physiology and Centre for Advanced Discovery and Experimental Therapeutics, Division of Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester Academic Health Science Centre, 3.24 Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, United Kingdom. +44 161 275 7969 [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/248187; this version posted January 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Heart failure is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for heart failure are worse than many common cancers and have not improved over time.
    [Show full text]
  • Dual Activation of Phosphodiesterase 3 and 4 Regulates Basal Cardiac Pacemaker Function and Beyond
    International Journal of Molecular Sciences Review Dual Activation of Phosphodiesterase 3 and 4 Regulates Basal Cardiac Pacemaker Function and Beyond Tatiana M. Vinogradova * and Edward G. Lakatta Laboratory of Cardiovascular Science, Intramural Research Program, National Institute on Aging, National Institute of Health, 251 Bayview Boulevard, Baltimore, MD 21224, USA; [email protected] * Correspondence: [email protected] Abstract: The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide Citation: Vinogradova, T.M.; Lakatta, evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the E.G.
    [Show full text]
  • Cardiac Camp-PKA Signaling Compartmentalization in Myocardial Infarction
    cells Review Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Infarction Anne-Sophie Colombe and Guillaume Pidoux * INSERM, UMR-S 1180, Signalisation et Physiopathologie Cardiovasculaire, Université Paris-Saclay, 92296 Châtenay-Malabry, France; [email protected] * Correspondence: [email protected] Abstract: Under physiological conditions, cAMP signaling plays a key role in the regulation of car- diac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (also known as GPCRs) with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocar- dial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it Citation: Colombe, A.-S.; Pidoux, G. appears to be profoundly impaired. Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Keywords: heart; myocardial infarction; cardiomyocytes; cAMP signaling; A-kinase anchoring Infarction.
    [Show full text]
  • Inhibition of Phosphodiesterase 11 (PDE11) Impacts on Sperm Quality
    International Journal of Impotence Research (2005) 17, 385–386 & 2005 Nature Publishing Group All rights reserved 0955-9930/05 $30.00 www.nature.com/ijir Letter to the Editor Inhibition of phosphodiesterase 11 (PDE11) impacts on sperm quality Comment on ‘High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodies- terase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients.’ by Weeks JL, Zoraghi R, Beasley A, Sekhar KR, Francis SH, Corbin JD. (Int J Impot Res 2004 Nov 11 [Epub ahead of print]). G Pomara1* and G Morelli1 1Section of Urology, Department of Surgery, S Chiara Hospital, Pisa University, Italy International Journal of Impotence Research (2005) 17, 385–386. doi:10.1038/sj.ijir.3901304 There is a growing interest worldwide in under- tadalafil is a nonspecific PDE11A4 inhibitor, we believe standing potential crossreaction of PDE5 inhibitors that for safety purpose we should consider the possible with other proteins and we agree with the authors crossreaction with all the four human PDE11 variants. that PDE11 is one of the obvious candidates, since it This global evaluation may provide the highest thera- is closely related to the PDE5. Weeks et al should be peutic benefits/undesirable side effects ratio. For these congratulated because, comparing the potencies of reasons, we do not believe that all safety issues on tadalafil, vardenafil and sildenafil for PDE11A4 and tadalafil crossreaction with PDE11 are satisfied. the fold selectivity of these inhibitors for PDE5A1 Within human PDE11A family, the alternatively over PDE11A4, they provide several important spliced isoforms display unique tissue expression information for safety issues.
    [Show full text]
  • Phosphodiesterase-9 (PDE9) Inhibition with BAY 73-6691 Increases Corpus Cavernosum Relaxations Mediated by Nitric Oxide–Cyclic GMP Pathway in Mice
    International Journal of Impotence Research (2012) 25, 69–73 & 2012 Macmillan Publishers Limited All rights reserved 0955-9930/12 www.nature.com/ijir ORIGINAL ARTICLE Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide–cyclic GMP pathway in mice FH da Silva1, MN Pereira1, CF Franco-Penteado2, G De Nucci1, E Antunes1 and MA Claudino3 Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg À 1 per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration–responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group.
    [Show full text]
  • (PDE9): Chances and Challenges Against Neurodegeneration
    pharmaceuticals Review A Perspective on Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against Neurodegeneration Giovanni Ribaudo * , Maurizio Memo and Alessandra Gianoncelli Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy; [email protected] (M.M.); [email protected] (A.G.) * Correspondence: [email protected]; Tel.: +39-030-3717419 Abstract: As life expectancy increases, dementia affects a growing number of people worldwide. Be- sides current treatments, phosphodiesterase 9 (PDE9) represents an alternative target for developing innovative small molecules to contrast neurodegeneration. PDE inhibition promotes neurotransmitter release, amelioration of microvascular dysfunction, and neuronal plasticity. This review will provide an update on natural and nature-inspired PDE9 inhibitors, with a focus on the structural features of PDE9 that encourage the development of isoform-selective ligands. The expression in the brain, the presence within its structure of a peculiar accessory pocket, the asymmetry between the two subunits composing the protein dimer, and the selectivity towards chiral species make PDE9 a suitable target to develop specific inhibitors. Additionally, the world of natural compounds is an ideal source for identifying novel, possibly asymmetric, scaffolds, and xanthines, flavonoids, neolignans, and their derivatives are currently being studied. In this review, the available literature data were interpreted and clarified, from a structural point of view, taking advantage of molecular modeling: 3D structures of ligand-target complexes were retrieved, or built, and discussed. Citation: Ribaudo, G.; Memo, M.; Gianoncelli, A. A Perspective on Keywords: PDE9; Alzheimer’s disease; natural compounds; cGMP; neurodegeneration; xanthines Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against 1.
    [Show full text]
  • Phosphodiesterase 1 (Pde1) at the Crossroads of Calcium And
    PHOSPHODIESTERASE 1 (PDE1) AT THE CROSSROADS OF CALCIUM AND CYCLIC NUCLEOTIDE SIGNALING IN DIABETIC NEPHROPATHY by Asim Bikash Dey A Dissertation Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Biology Indianapolis, Indiana May 2020 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Simon J. Atkinson Department of Biology Dr. Mark C. Kowala Eli Lilly and Company Dr Ruben C. Aguilar Purdue University Dr. Guoli Dai Department of Biology Dr. Anthony J. Baucum II Department of Biology Approved by: Dr. Theodore R. Cummins Head of the Graduate Program 2 Dedicated to my family 3 ACKNOWLEDGMENTS I greatly appreciate Dr. Simon Atkinson at IUPUI and Dr. Mark Kowala at Eli Lilly and Company, my supervisors, for their support and encouragement throughout my graduate work in parallel to my day job. Their unrelenting support and profound belief in me have been instrumental in completing this work. Special thanks to Dr. Kowala for his flexibility that made it easier to juggle graduate work and the demands of a full-time job. I’m deeply indebted to Dr. Mark Rekhter whose unconditional support at all stages of my thesis made this a reality. Your insightful comments and input have helped me develop my critical analytical skills and help me become a better scientist. Completion of my experiments would not have been possible without your critical input, technical assistance and constant support over the last several years. I also would like to thank my committee members, Dr. Guoli Dai, Dr.
    [Show full text]
  • PDE1C Deficiency Antagonizes Pathological Cardiac Remodeling and Dysfunction
    PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction Walter E. Knighta,b, Si Chena,b, Yishuai Zhanga, Masayoshi Oikawaa, Meiping Wua,c, Qian Zhoua, Clint L. Millera, Yujun Caia, Deanne M. Mickelsena, Christine Moravecd, Eric M. Smalla, Junichi Abea, and Chen Yana,1 aAab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14641; bDepartment of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14641; cDepartment of Cardiology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China 201203; and dDepartment of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH 44195 Edited by Joseph A. Beavo, University of Washington School of Medicine, Seattle, WA, and approved September 20, 2016 (received for review May 13, 2016) Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major signaling modules have distinct, unique, and often even opposing phosphodiesterase activity in human myocardium, but its function physiological roles. in the heart remains unknown. Using genetic and pharmacological Cyclic nucleotide phosphodiesterases (PDEs), by catalyzing approaches, we studied the expression, regulation, function, and cyclic nucleotide hydrolysis, play a critical role in regulating the underlying mechanisms of PDE1C in the pathogenesis of cardiac amplitude, duration, and compartmentalization of cyclic nucleotide remodeling and dysfunction. PDE1C expression is up-regulated in signaling. PDEs constitute a superfamily of enzymes with 22 dif- mouse and human failing hearts and is highly expressed in cardiac ferent genes and more than 100 mRNAs grouped into 11 broad myocytes but not in fibroblasts. In adult mouse cardiac myocytes, families (PDE1–PDE11) based on distinct structural, kinetic, reg- PDE1C deficiency or inhibition attenuated myocyte death and ulatory, and inhibitory properties.
    [Show full text]