Assembly, Operation, and Disassembly of Protein Complexes
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The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (Cad) by Phosphorylation and Protein-Protein Interactions
THE REGULATION OF CARBAMOYL PHOSPHATE SYNTHETASE-ASPARTATE TRANSCARBAMOYLASE-DIHYDROOROTASE (CAD) BY PHOSPHORYLATION AND PROTEIN-PROTEIN INTERACTIONS Eric M. Wauson A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2007 Approved by: Lee M. Graves, Ph.D. T. Kendall Harden, Ph.D. Gary L. Johnson, Ph.D. Aziz Sancar M.D., Ph.D. Beverly S. Mitchell, M.D. 2007 Eric M. Wauson ALL RIGHTS RESERVED ii ABSTRACT Eric M. Wauson: The Regulation of Carbamoyl Phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (CAD) by Phosphorylation and Protein-Protein Interactions (Under the direction of Lee M. Graves, Ph.D.) Pyrimidines have many important roles in cellular physiology, as they are used in the formation of DNA, RNA, phospholipids, and pyrimidine sugars. The first rate- limiting step in the de novo pyrimidine synthesis pathway is catalyzed by the carbamoyl phosphate synthetase II (CPSase II) part of the multienzymatic complex Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, Dihydroorotase (CAD). CAD gene induction is highly correlated to cell proliferation. Additionally, CAD is allosterically inhibited or activated by uridine triphosphate (UTP) or phosphoribosyl pyrophosphate (PRPP), respectively. The phosphorylation of CAD by PKA and ERK has been reported to modulate the response of CAD to allosteric modulators. While there has been much speculation on the identity of CAD phosphorylation sites, no definitive identification of in vivo CAD phosphorylation sites has been performed. Therefore, we sought to determine the specific CAD residues phosphorylated by ERK and PKA in intact cells. -
Maturation of Sucrase-Isomaltase Complex in Human Fetal Small and Large Intestine During Gestation
136 TRIADOU AND ZWEIBAUM 003 1-3998/85/190 1-0 136$02.0010 PEDIATRIC RESEARCH Vol. 19, No. 1, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc Prin~edin U.S. A. Maturation of Sucrase-Isomaltase Complex in Human Fetal Small and Large Intestine during Gestation NICOLE TRIADOU AND ALAIN ZWEIBAUM UnitP de Recherches de GPnetique MPdicale [N. T.], INSERM U 12, H6pital des Enfants Malades, and Unite de Recherches sur le MPtabolisme et la Diffirmciation des Cellules en Culture [A.Z.],INSERM U 178, H6pital Broussais, Paris, France ABSTRACT. Sucrase-isomaltase complex is expressed in chromatography. The homogeneity of the preparation was as- human small intestine throughout gestation and in the large sessed by polyacrylamide gel electrophoresis and crossed immu- intestine between 12 and 30 wk. The molecular form of the noelectrophoresis against an antihuman brush-border antiserum enzyme was studied in the brush-border membrane frac- (12). Rabbits were immunized by sc injections at 15-day intervals tions by the immunoblotting method. Before 30 wk of with 0.2 mg of purified enzyme and bled 7 days after the fourth gestation, the enzyme is present only as the high molecular injection. Immunoglobulins G were prepared by ion exchange weight prosucrase-isomaltase, while from 30 wk until birth chromatography (10, 15). the two subunits are also present. The fetal enzyme, as its Brush-border separation, transjer, and identification of antigen. proform and as its two subunits, has a faster mobility in Preparations of the brush-border fractions of small and large sodium-dodecylsulfate polyacrylamide gel electrophoresis, intestine were obtained by the calcium precipitation method as than the adult enzyme (removal of sialic acid residues from previously described (12). -
Using Chemical Inhibitors to Probe AAA Protein Conformational Dynamics and Cellular Functions Steinman and Kapoor 47
Available online at www.sciencedirect.com ScienceDirect Using chemical inhibitors to probe AAA protein conformational dynamics and cellular functions Jonathan B Steinman and Tarun M Kapoor The AAA proteins are a family of enzymes that play key roles in shRNA) can often take longer than many of the cellular diverse dynamic cellular processes, ranging from proteostasis processes they are involved in and thereby lead to the to directional intracellular transport. Dysregulation of AAA accumulation of phenotypes not directly linked to their proteins has been linked to several diseases, including cancer, functions. Destabilization of multi-protein complexes also suggesting a possible therapeutic role for inhibitors of these has the potential to cause dominant negative effects. Simi- enzymes. In the past decade, new chemical probes have been larly, the slowly or non-hydrolyzable ATP analogs often used developed for AAA proteins including p97, dynein, midasin, and to study AAA proteins in vitro are poorly suited for studying ClpC1. In this review, we discuss how these compounds have these enzymes in cellular contexts, as they are generally been used to study the cellular functions and conformational unable to cross cell membranes and tend to inhibit multiple dynamics of AAA proteins. We discuss future directions for different enzymes. inhibitor development and early efforts to utilize AAA protein inhibitors in the clinical setting. Cell-permeable chemical inhibitors of AAA proteins have the potential to overcome many of these challenges. They Address can act on timescales that match the processes driven by Laboratory of Chemistry and Cell Biology, Rockefeller University, New these enzymes, limiting the degree to which cells can York, United States activate compensatory pathways or accumulate indirect Corresponding author: Kapoor, Tarun M ([email protected]) effects. -
Allostery and Cooperativity in Multimeric Proteins: Bond-To-Bond Propensities in Atcase
Allostery and cooperativity in multimeric proteins: bond-to-bond propensities in ATCase Maxwell Hodges1,3, Mauricio Barahona2,3, and Sophia N. Yaliraki1,3 1Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom 2Department of Mathematics, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom 3Institute of Chemical Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom September 30, 2019 Abstract Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. -
A Double-Hexameric MCM2-7 Complex Is Loaded Onto Origin DNA During Licensing of Eukaryotic DNA Replication
A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication Cecile Evrina, Pippa Clarkea, Juergen Zecha, Rudi Lurzb, Jingchuan Sunc, Stefan Uhlea,1, Huilin Lic, Bruce Stillmand,2, and Christian Specka,2 aDNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom; bMicroscopy Unit, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany; cBiology Department, Brookhaven National Laboratory, Upton, NY 11973; and dCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 Contributed by Bruce Stillman, October 6, 2009 (sent for review September 22, 2009) During pre-replication complex (pre-RC) formation, origin recog- to form the pre-initiation complex (pre-IC). Binding of pre-IC nition complex (ORC), Cdc6, and Cdt1 cooperatively load the proteins and protein kinase activity stimulates MCM2-7 helicase 6-subunit mini chromosome maintenance (MCM2-7) complex onto activity (13). Pre-IC formation culminates in the recruitment of DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that DNA polymerases and the start of active DNA replication (14). restricts DNA replication to once per cell cycle. During S phase ORC and Cdc6 belong to the AAAϩ family of ATP binding MCM2-7 functions as part of the replicative helicase but within the proteins (4, 15), a family that commonly forms ring- or spiral- pre-RC MCM2-7 is inactive. The organization of replicative DNA shaped structures. A spiral-shaped structure consisting of 5 helicases before and after loading onto DNA has been studied in AAAϩ proteins within the replication factor C (RFC) complex bacteria and viruses but not eukaryotes and is of major importance functions to destabilize the homotrimeric proliferating cell nu- for understanding the MCM2-7 loading mechanism and replisome clear antigen (PCNA) ring during PCNA loading onto DNA. -
Metabolomics-Assisted Proteomics Identifies Succinylation and SIRT5 As Important Regulators of Cardiac Function
Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function Sushabhan Sadhukhana, Xiaojing Liub,c,d, Dongryeol Ryue, Ornella D. Nelsona, John A. Stupinskif, Zhi Lia, Wei Cheng, Sheng Zhangg, Robert S. Weissf, Jason W. Locasaleb,c,d, Johan Auwerxe,1, and Hening Lina,h,1 aDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853; bDuke Cancer Institute, Duke University School of Medicine, Durham, NC 27710; cDuke Molecular Physiology Institute, Duke University School of Medicine, Durham, NC 27710; dDepartment of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC 27710; eLaboratory of Integrative and Systems Physiology, School of Life Sciences, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; fDepartment of Biomedical Sciences, Cornell University, Ithaca, NY 14853; gProteomics & Mass Spectrometry Facility, Institute of Biotechnology, Cornell University, Ithaca, NY 14853; and hHoward Hughes Medical Institute, Cornell University, Ithaca, NY 14853 Edited by Kevan M. Shokat, University of California, San Francisco, CA, and approved March 9, 2016 (received for review October 7, 2015) Cellular metabolites, such as acyl-CoA, can modify proteins, leading SIRT4 and SIRT5 have very weak deacetylase activities (14). SIRT5 to protein posttranslational modifications (PTMs). One such PTM is possesses unique enzymatic activity on hydrolyzing negatively lysine succinylation, which is regulated by sirtuin 5 (SIRT5). Although charged -
Sequence Analysis of the AAA Protein Family
Prorein Science (1997), 62043-2058. Cambridge University Press. Printed in the USA. Copyright 0 1997 The Protein Society REVIEW Sequence analysis of the AAA protein family ANDREAS BEYER Institut fur Physiologische Chemie, Medizinische Fakultat, Ruhr-Universitat. D-44780 Bochum, Germany (RECEIVEDMarch 12, 1997; ACCEPTEDJune 27, 1997) Abstract The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside thecanonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or evenno similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases. Keywords: AAA family; ATPase; evolution; secondary structure prediction; Walker family The AAA protein family (“triple-A family”) is a group of ATPases SF 6, respectively (see Table 1). -
A 3.5-A Resolution Study (Protein Crystallography/Enzyme Activation) J
Proc. NatL Acad. Sci. USA Vol. 79, pp. 3125-3128, May 1982 Biochemistry Gross quaternary changes in aspartate carbamoyltransferase are induced by the binding of N-(phosphonacetyl)-L-aspartate: A 3.5-A resolution study (protein crystallography/enzyme activation) J. E. LADNER, J. P. KITCHELL, R. B. HONZATKO, H. M. KE, K. W. VoLZ, A. J. KALB (GILBOA)t, R. C. LADNER, AND W. N. LIPSCOMB Gibbs Chemical Laboratory, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138 Contributed by W. N. Lipscomb, February 11, 1982 ABSTRACT The three-dimensional structure of the complex of N-(phosphonacetyl)-L-aspartate with aspartate carbamoyl- transferase (carbamoylphosphate:L-aspartate carbamoyltransfer- ase, EC 2.1.3.2) has been determined to a nominal resolution of 3.5 A by single-crystal x-ray diffraction methods. Initial phases were obtained by the method of "molecular tectonics": beginning with the structure ofthe CTP-protein complex, the domains ofthe catalytic and regulatory chains were manipulated as separate rigid bodies. The resulting coordinates were used to calculate an elec- tron density map, which was then back transformed to give a set of calculated amplitudes and phases. Using all observed data, we obtained a crystallographic R factor between observed and cal- culated amplitudes F. and F, of 0.46. An envelope was then ap- A B plied to a 2F. - Fc map and the density was averaged across the axis. Two of an R factor molecular twofold cycles averaging yielded FIG. 1. Schematic drawings of the aspartate carbamoyltransfer- of0.25. In this complex, we find that the twocatalytic trimers have ase molecule. -
Using Peptides to Examine the Interaction Interface Between Aspartate Transcarbamoylase and Dihydroorotase in Pyrimidine Biosynthesis in Aquifex Aeolicus Nouf Alyami
Eastern Michigan University DigitalCommons@EMU Master's Theses, and Doctoral Dissertations, and Master's Theses and Doctoral Dissertations Graduate Capstone Projects 7-14-2015 Using peptides to examine the interaction interface between Aspartate transcarbamoylase and Dihydroorotase in pyrimidine biosynthesis in Aquifex aeolicus Nouf Alyami Follow this and additional works at: http://commons.emich.edu/theses Part of the Chemistry Commons Recommended Citation Alyami, Nouf, "Using peptides to examine the interaction interface between Aspartate transcarbamoylase and Dihydroorotase in pyrimidine biosynthesis in Aquifex aeolicus" (2015). Master's Theses and Doctoral Dissertations. 641. http://commons.emich.edu/theses/641 This Open Access Thesis is brought to you for free and open access by the Master's Theses, and Doctoral Dissertations, and Graduate Capstone Projects at DigitalCommons@EMU. It has been accepted for inclusion in Master's Theses and Doctoral Dissertations by an authorized administrator of DigitalCommons@EMU. For more information, please contact [email protected]. Using Peptides to Examine the Interaction Interface between Aspartate transcarbamoylase and Dihydroorotase in Pyrimidine Biosynthesis in Aquifex aeolicus by Nouf Alyami Thesis Submitted to the Department of Chemistry Eastern Michigan University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Chemistry Thesis Committee: Hedeel Evans, Ph.D., Chair Deborah Heyl-Clegg, Ph.D. Vance Kennedy, Ph.D. July 14, 2015 Ypsilanti, Michigan ii DEDICATION This work is dedicated to my parents, without whose support it would not have been possible. A special feeling of gratitude to my loving siblings Nada, Bayan, and Bader whose laughter is like music to my soul. Also, I dedicate this work to my two grandmothers whose hearts are full of prayers for me. -
Maple Syrup Urine Disease
Maple syrup urine disease Description Maple syrup urine disease is an inherited disorder in which the body is unable to process certain protein building blocks (amino acids) properly. The condition gets its name from the distinctive sweet odor of affected infants' urine. It is also characterized by poor feeding, vomiting, lack of energy (lethargy), abnormal movements, and delayed development. If untreated, maple syrup urine disease can lead to seizures, coma, and death. Maple syrup urine disease is often classified by its pattern of signs and symptoms. The most common and severe form of the disease is the classic type, which becomes apparent soon after birth. Variant forms of the disorder become apparent later in infancy or childhood and are typically milder, but they still lead to delayed development and other health problems if not treated. Frequency Maple syrup urine disease affects an estimated 1 in 185,000 infants worldwide. The disorder occurs much more frequently in the Old Order Mennonite population, with an estimated incidence of about 1 in 380 newborns. Causes Mutations in the BCKDHA, BCKDHB, and DBT genes can cause maple syrup urine disease. These three genes provide instructions for making proteins that work together as part of a complex. The protein complex is essential for breaking down the amino acids leucine, isoleucine, and valine, which are present in many kinds of food, particularly protein-rich foods such as milk, meat, and eggs. Mutations in any of these three genes reduce or eliminate the function of the protein complex, preventing the normal breakdown of leucine, isoleucine, and valine. -
New Mesh Headings for 2018 Single Column After Cutover
New MeSH Headings for 2018 Listed in alphabetical order with Heading, Scope Note, Annotation (AN), and Tree Locations 2-Hydroxypropyl-beta-cyclodextrin Derivative of beta-cyclodextrin that is used as an excipient for steroid drugs and as a lipid chelator. Tree locations: beta-Cyclodextrins D04.345.103.333.500 D09.301.915.400.375.333.500 D09.698.365.855.400.375.333.500 AAA Domain An approximately 250 amino acid domain common to AAA ATPases and AAA Proteins. It consists of a highly conserved N-terminal P-Loop ATPase subdomain with an alpha-beta-alpha conformation, and a less-conserved C- terminal subdomain with an all alpha conformation. The N-terminal subdomain includes Walker A and Walker B motifs which function in ATP binding and hydrolysis. Tree locations: Amino Acid Motifs G02.111.570.820.709.275.500.913 AAA Proteins A large, highly conserved and functionally diverse superfamily of NTPases and nucleotide-binding proteins that are characterized by a conserved 200 to 250 amino acid nucleotide-binding and catalytic domain, the AAA+ module. They assemble into hexameric ring complexes that function in the energy-dependent remodeling of macromolecules. Members include ATPASES ASSOCIATED WITH DIVERSE CELLULAR ACTIVITIES. Tree locations: Acid Anhydride Hydrolases D08.811.277.040.013 Carrier Proteins D12.776.157.025 Abuse-Deterrent Formulations Drug formulations or delivery systems intended to discourage the abuse of CONTROLLED SUBSTANCES. These may include physical barriers to prevent chewing or crushing the drug; chemical barriers that prevent extraction of psychoactive ingredients; agonist-antagonist combinations to reduce euphoria associated with abuse; aversion, where controlled substances are combined with others that will produce an unpleasant effect if the patient manipulates the dosage form or exceeds the recommended dose; delivery systems that are resistant to abuse such as implants; or combinations of these methods. -
Download 20190410); Fragmentation for 20 S
ARTICLE https://doi.org/10.1038/s41467-020-17387-y OPEN Multi-layered proteomic analyses decode compositional and functional effects of cancer mutations on kinase complexes ✉ Martin Mehnert 1 , Rodolfo Ciuffa1, Fabian Frommelt 1, Federico Uliana1, Audrey van Drogen1, ✉ ✉ Kilian Ruminski1,3, Matthias Gstaiger1 & Ruedi Aebersold 1,2 fi 1234567890():,; Rapidly increasing availability of genomic data and ensuing identi cation of disease asso- ciated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are trans- lated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are asso- ciated with topological changes and affected phosphorylation of known cancer driver pro- teins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions. 1 Department of Biology, Institute of Molecular Systems Biology, ETH Zurich,