Myristoylation-Dependent Replication and Assembly of Human

Total Page:16

File Type:pdf, Size:1020Kb

Myristoylation-Dependent Replication and Assembly of Human Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 523-527, January 1990 Biochemistry Myristoylation-dependent replication and assembly of human immunodeficiency virus 1 (AIDS/virus assembly/gag precursor) MARTIN BRYANT*t AND LEE RATNERtf§ Departments of *Pediatrics, tMedicine, and §Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110 Communicated by Stuart Kornfeld, September 25, 1989 (receivedfor review May 23, 1989) ABSTRACT Covalent linkage of myristic acid to the N- virus types I and II, human immunodeficiency virus 1 (HIV-1), terminal glycine residue ofPr55gag, the precursor ofthe major and simian immunodeficiency virus type I] has recently been structural proteins of human immunodeficiency virus 1 (HIV- described (1, 18-23). In cells infected by MoMLV (22) or 1), facilitates an essential step in virus assembly and propaga- MPMV (23), myristoylation of the gag polypeptide is required tion. Substitution of the myristoyl-acceptor glycine with ala- for normal virion assembly and infectious particle production. nine, in a functional clone of HIV-1, eliminates virus replica- HIV, like other mammalian retroviruses, encodes a gag tion. Complementation of this defect, in trans, restores precursor, Pr55gag, which is normally processed by the viral infectious particle production. The nonmyristoylated (myr-) protease to the major structural proteins found in the mature gag precursor accumulates in infected cells and is not processed extracellular virus particle. Both Pr55gag and the matrix into the mature capsid components of the intact virion. How- protein (p17), which is proteolytically cleaved from the ever, myr- Pr55gag can be processed by purified HIV protease N-terminal end ofPr55gag, are myristoylated (see Fig. 1) (24, in vitro, demonstrating that the myristoyl moiety is not required 25). Analysis of the amino acid sequence of these proteins for cleavage by the protease. Myristoylation of Pr55gag is not from different HIV-1 isolates shows that the myristoylation necessary for localization but is required for stable membrane acceptor is always an N-terminal glycine residue (26-28). association and assembly of HIV-1. Furthermore, the Prl80gag-pol precursor, which contains gag proteins as well as the viral replicative enzymes [prote- The covalent attachment of long-chain fatty acids to select ase, reverse transcriptase (RT), and integrase], is probably subsets of eukaryotic cellular proteins and viral polypeptides also myristoylated. The current study examines the role of confers a change in hydrophobicity that may be important in myristoylation of the structural gag polyprotein precursor defining protein quaternary structure, directing intracellular Pr55gag of HIV-1 in virus replication. Our results show that trafficking and signal transduction, and/or in specific pro- addition of the myristate moiety is required for stable mem- tein-protein or protein-lipid interactions at the plasma mem- brane association, proteolytic processing, and assembly of brane. The specific fatty acid and its distinct mode of Pr55gag into infectious extracellular particles of HIV-1. attachment probably influence the unique biochemical func- tion served by each acylated protein (1-5). MATERIALS AND METHODS The most thoroughly studied acyl proteins have been Cell Lines. T-lymphoid cell lines H9 and Jurkat were virus-related polypeptides because of their high level of provided by J. Hoxie and R. C. Gallo (National Cancer expression in infected cells. Schmidt and Schlesinger (6) were Institute). COS-1 and HeLa cell lines were obtained from the first to describe the attachment of fatty acid to the envelope American Type Culture Collection. glycoproteins of vesicular stomatitis and Sindbis viruses and DNA Clones. pGG1 contains a functional clone of HIV-1 to suggest an association of this type of protein modification and was previously designated pHXB2gpt2 (29). Plasmid with virus assembly and propagation (7). Subsequently, the pAenv is a derivative of pGG1 constructed by digestion with structural proteins of nonenveloped viruses have also been the restriction endonucleases Sal I and Xho I, followed by shown to contain covalently bound fatty acid. The capsid treatment with T4 DNA ligase thus deleting the entire enve- protein VP4 and its precursors in picornaviruses are myris- lope gene of HIV-1 (nucleotides 5366-8474). toylated (8-11). The position of the myristate moiety at the Mutagenesis. The glycine to alanine substitution was made interface between protein subunits in poliovirus suggests that with the 20-mer oligonucleotide GCTCTCGCAGCCATCTC- it may play a role in virus capsid assembly (12). The structural TCT using oligonucleotide-directed mutagenesis as de- proteins ofpolyomavirus (10) and simian virus 40 (11), as well scribed by Kunkel (30). The resultant plasmid was designated as the PreS1 protein ofhepadnaviruses (i.e., hepatitis B virus) pGA1 and the mutation was confirmed by the dideoxynucle- (13), are also modified by attachment of myristic acid and otide chain-termination method for sequencing DNA (31). may influence nucleocapsid assembly and/or infectivity. Transfection. Cells were transfected by the calcium phos- Myristoylation of p6Ov-src of Rous sarcoma virus is re- phate precipitation technique for gene transfer (32). Stable quired for its stable association with cellular membranes and transfectants were isolated by cotransfection of HeLa cells morphological transformation of cells (14, 15). Similarly, with either pGA1 or pGG1 and SFneo [similar to pSV2neo myristoylation has been proposed to be an important factor in with the addition of the spleen focus-forming virus long transformation by gag-onc fusion proteins with kinase activity terminal repeat (33)]. Cell clones resistant to G418 (400 (16, 17). In addition, fatty acid modification of the gag pre- ,ug/ml, GIBCO) and positive by HIV-1 p24 ELISA (DuPont) cursor to the internal structural proteins of many mammalian were selected for protein studies. retroviruses [Moloney murine leukemia virus (MoMLV), Ma- son-Pfizer monkey virus (MPMV), human T-cell leukemia Abbreviations: MoMLV, Moloney murine leukemia virus; MPMV, Mason-Pfizer monkey virus; HIV, human immunodeficiency virus; RT, reverse transcriptase. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Washington payment. This article must therefore be hereby marked "advertisement" University School of Medicine, Clinical Science Building Box 8125, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 660 South Euclid Avenue, Saint Louis, MO 63110. 523 Downloaded by guest on September 26, 2021 524 Biochemistry: Bryant and Ratner Proc. Natl. Acad. Sci. USA 87 (1990) Virus Assays. Cell supernatants were tested on various Biological Effect of Myristoylation Minus Mutation. The days posttransfection for virus-specific antigen using the p24 pGA1 mutant and the parent plasmid pGG1 were transfected ELISA (sensitivity of 0.03 ng/ml). Both the major structural into CD4- HeLa (Fig. 2) or COS-1 cells (not shown) using nucleocapsid protein of HIV-1, p24, and the p24-containing calcium phosphate coprecipitation (32). On day 4 posttrans- Pr55gag can be detected by this assay (unpublished obser- fection, CD4' human lymphoid cells (H9) were added to vation). Virions were concentrated 10:1 from culture super- allow propagation and amplification of infectious virus re- natants by polyethylene glycol precipitation (30% in 150 mM leased from the transfected cells. The nonadherent H9 cells NaCl/0.1 mM phenylmethylsulfonyl fluoride), solubilized, were removed from the adherent HeLa cells on day 8 and and virus-associated RT activity was measured by [32P]TTP were maintained in culture for an additional 2 weeks. Virus- incorporation using a poly(rA)-oligo(dT) template (34). specific antigen production, as measured by p24 antigen Immunoprecipitation and Immunoblotting. HIV-1-specific ELISA, was detected in supernatant solutions from both antigens in lysates from 4 x 106 cells or cell-free supernatants pGG1 and pGA1 transfected cultures prior to the addition of were characterized by immunoblot techniques (35) or were the H9 cells (Fig. 2). However, neither virus antigen nor RT immunoprecipitated after labeling with [35S]methionine/ activity could be detected in the H9 cultures after cocultiva- cysteine (specific activity, 1031 Ci/mmol; 1 Ci = 37 GBq) or tion with pGA1 transfected HeLa (Fig. 2) or COS-1 cells (not [3H]myristic acid (39.3 Ci/mmol). Cells labeled with myris- shown). In contrast, production of infectious HIV by the tate were grown in 5% delipidated fetal calf serum. Pooled pGG1 transfected cells was readily identified by syncytia AIDS patients' sera containing a high titer of antibody to HIV formation among the H9 cells, p24 soluble antigen produc- antigens were used in both procedures. Cell pellets were tion, and RT activity (Fig. 2). The pGA1 defect could, how- lysed in RIPA buffer [50 mM Tris-HCI, pH 7.4/0.5% Triton ever, be complemented in trans, by the gag-pol expression X-100/100 mM NaCl/aprotinin (100 units/ml)/0.1 mM phe- plasmid pAenv (Fig. 2). Similar results were obtained after nylmethylsulfonyl fluoride] and used directly for immunoblot direct transfection of the CD4+ Jurkat cell line (data not analysis. The HIV-1 and cellular proteins were separated by shown). These results show that the glycine to alanine sub- SDS/PAGE (36) and transferred by electroblotting to a stitution at the N terminus of the gag polyprotein Pr55gag
Recommended publications
  • LPS Induces GFAT2 Expression to Promote O-Glcnacylation
    1 LPS induces GFAT2 expression to promote O-GlcNAcylation and 2 attenuate inflammation in macrophages 3 4 5 Running title: GFAT2 is a FoxO1-dependent LPS-inducible gene 6 7 Hasanain AL-MUKH, Léa BAUDOIN, Abdelouhab BOUABOUD, José-Luis SANCHEZ- 8 SALGADO, Nabih MARAQA, Mostafa KHAIR, Patrick PAGESY, Georges BISMUTH, 9 Florence NIEDERGANG and Tarik ISSAD 10 11 Université de Paris, Institut Cochin, CNRS, INSERM, F-75014 Paris, France 12 Address correspondence to: Tarik Issad, Institut Cochin, Department of Endocrinology, 13 Metabolism and Diabetes, 24 rue du Faubourg Saint-Jacques, 75014 Paris FRANCE. Tel : + 33 1 14 44 41 25 67; E-mail: tarik.issad@inserm.fr 15 16 1 1 Abstract 2 O-GlcNAc glycosylation is a reversible post-translational modification that regulates the 3 activity of intracellular proteins according to glucose availability and its metabolism through 4 the hexosamine biosynthesis pathway (HBP). This modification has been involved in the 5 regulation of various immune cell types, including macrophages. However, little is known 6 concerning the mechanisms that regulate protein O-GlcNAcylation level in these cells. In the 7 present work, we demonstrate that LPS treatment induces a marked increase in protein O- 8 GlcNAcylation in RAW264.7 cells, bone-marrow-derived and peritoneal mouse 9 macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in 10 macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine 11 production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced 12 by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with 13 an increased expression and activity of glutamine fructose 6-phosphate amido-transferase 14 (GFAT), the enzyme that catalyzes the rate-limiting step of the HBP.
    [Show full text]
  • A Role for N-Myristoylation in Protein Targeting
    A Role for N-Myristoylation in Protein Targeting: NADH-Cytochrome b5 Reductase Requires Myristic Acid for Association with Outer Mitochondrial But Not ER Membranes Nica Borgese,** Diego Aggujaro,* Paola Carrera, ~ Grazia Pietrini,* and Monique Bassetti* *Consiglio Nazionale deUe Ricerche Cellular and Molecular Pharmacology Center, Department of Pharmacology, University of Milan, 20129 Milan, Italy; ~Faculty of Pharmacy, University of Reggio Calabria, 88021 Roccelletta di Borgia, Catanzaro, Italy; and ~Scientific Institute "San Raffaele," 20132 Milan, Italy Abstract. N-myristoylation is a cotranslational modifi- investigated by immunofluorescence, immuno-EM, and Downloaded from http://rupress.org/jcb/article-pdf/135/6/1501/1267948/1501.pdf by guest on 27 September 2021 cation involved in protein-protein interactions as well cell fractionation. By all three techniques, the wt pro- as in anchoring polypeptides to phospholipid bilayers; tein localized to ER and mitochondria, while the non- however, its role in targeting proteins to specific subcel- myristoylated mutant was found only on ER mem- lular compartments has not been clearly defined. The branes. Pulse-chase experiments indicated that this mammalian myristoylated flavoenzyme NADH-cyto- altered steady state distribution was due to the mu- chrome b 5 reductase is integrated into ER and mito- tant's inability to target to mitochondria, and not to its chondrial outer membranes via an anchor containing a enhanced instability in that location. Both wt and mu- stretch of 14 uncharged amino acids downstream to the tant reductase were resistant to Na2CO 3 extraction and NH2-terminal myristoylated glycine. Since previous partitioned into the detergent phase after treatment of studies suggested that the anchoring function could be a membrane fraction with Triton X-114, demonstrating adequately carried out by the 14 uncharged residues, that myristic acid is not required for tight anchoring of we investigated a possible role for myristic acid in re- reductase to membranes.
    [Show full text]
  • RNF11 at the Crossroads of Protein Ubiquitination
    biomolecules Review RNF11 at the Crossroads of Protein Ubiquitination Anna Mattioni, Luisa Castagnoli and Elena Santonico * Department of Biology, University of Rome Tor Vergata, Via della ricerca scientifica, 00133 Rome, Italy; Anna.Mattioni@gmail.com (A.M.); Castagnoli@uniroma2.it (L.C.) * Correspondence: Elena.Santonico@uniroma2.it Received: 29 September 2020; Accepted: 8 November 2020; Published: 11 November 2020 Abstract: RNF11 (Ring Finger Protein 11) is a 154 amino-acid long protein that contains a RING-H2 domain, whose sequence has remained substantially unchanged throughout vertebrate evolution. RNF11 has drawn attention as a modulator of protein degradation by HECT E3 ligases. Indeed, the large number of substrates that are regulated by HECT ligases, such as ITCH, SMURF1/2, WWP1/2, and NEDD4, and their role in turning off the signaling by ubiquitin-mediated degradation, candidates RNF11 as the master regulator of a plethora of signaling pathways. Starting from the analysis of the primary sequence motifs and from the list of RNF11 protein partners, we summarize the evidence implicating RNF11 as an important player in modulating ubiquitin-regulated processes that are involved in transforming growth factor beta (TGF-β), nuclear factor-κB (NF-κB), and Epidermal Growth Factor (EGF) signaling pathways. This connection appears to be particularly significant, since RNF11 is overexpressed in several tumors, even though its role as tumor growth inhibitor or promoter is still controversial. The review highlights the different facets and peculiarities of this unconventional small RING-E3 ligase and its implication in tumorigenesis, invasion, neuroinflammation, and cancer metastasis. Keywords: Ring Finger Protein 11; HECT ligases; ubiquitination 1.
    [Show full text]
  • Every Detail Matters. That Is, How the Interaction Between Gα Proteins and Membrane Affects Their Function
    membranes Review Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function Agnieszka Polit * , Paweł Mystek and Ewa Błasiak Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; pawel.mystek@uj.edu.pl (P.M.); ewa.blasiak@uj.edu.pl (E.B.) * Correspondence: a.polit@uj.edu.pl; Tel.: +48-12-6646156 Abstract: In highly organized multicellular organisms such as humans, the functions of an individ- ual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein–lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interre- lated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions Citation: Polit, A.; Mystek, P.; within these proteins and interactions with other components of the heterocomplex.
    [Show full text]
  • Interaction of a G Protein with an Activated Receptor Opens the Interdomain Interface in the Alpha Subunit
    Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit Ned Van Epsa,1, Anita M. Preiningerb,1, Nathan Alexanderc,1, Ali I. Kayab, Scott Meierb, Jens Meilerc,2, Heidi E. Hammb,2, and Wayne L. Hubbella,2 aJules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-7008; bDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6600; and cDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232-6600 Contributed by Wayne L. Hubbell, April 14, 2011 (sent for review March 12, 2011) In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on the Gα subunit of a heterotrimeric G protein. In an initial step, receptor interaction with Gα acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechan- ism is unknown. In this study, site-directed spin labeling and double electron–electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of Gα, the extension of which is a high-affinity receptor binding element. signal transduction ∣ structural polymorphism he α-subunit (Gα) of heterotrimeric G proteins (Gαβγ) med- Tiates signal transduction in a variety of cell signaling pathways Fig. 1. Receptor activation of G proteins leads to a separation between (1).
    [Show full text]
  • Gi- and Gs-Coupled Gpcrs Show Different Modes of G-Protein Binding
    Gi- and Gs-coupled GPCRs show different modes of G-protein binding Ned Van Epsa, Christian Altenbachb,c, Lydia N. Caroa,1, Naomi R. Latorracad,e,f,g, Scott A. Hollingsworthd,e,f,g, Ron O. Drord,e,f,g, Oliver P. Ernsta,h,2, and Wayne L. Hubbellb,c,2 aDepartment of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; bStein Eye Institute, University of California, Los Angeles, CA 90095; cDepartment of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; dDepartment of Computer Science, Stanford University, Stanford, CA 94305; eDepartment of Structural Biology, Stanford University, Stanford, CA 94305; fDepartment of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; gInstitute for Computational and Mathematical Engineering, Stanford University, Stanford, CA 94305; and hDepartment of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada Contributed by Wayne L. Hubbell, January 17, 2018 (sent for review December 20, 2017; reviewed by David S. Cafiso and Thomas P. Sakmar) More than two decades ago, the activation mechanism for the differing only by specific side-chain interactions, or is there a membrane-bound photoreceptor and prototypical G protein-coupled specificity code in the receptor involving the allowed magnitude of receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes displacement of particular helices? Of considerable interest are in ligand–receptor interaction, movement of specific transmembrane GPCRs which couple to multiple G-protein subtypes and can helices within the receptor opens a crevice at the cytoplasmic surface, sample diverse conformational landscapes. allowing for coupling of heterotrimeric guanine nucleotide-binding In the present study, SDSL and double electron–electron reso- proteins (G proteins).
    [Show full text]
  • Synthesis and Investigation of Bacterial Effector Molecules
    Synthesis and investigation of bacterial effector molecules Michael Franz Albers Doctoral Thesis, Department of Chemistry Umeå University, 2016 Responsible publisher under swedish law: the Dean of the Faculty of Science and Technology This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-411-0 Electronic version available at http://umu.diva-portal.org/ Tryck/Printed by: VMC-KBC Umeå Umeå, Sweden, 2016 Table of Contents Table of Contents i Abstract iii List of Abbreviations iv List of Publications vii Author contributions viii Papers by the author, but not included in this thesis viii Enkel sammanfattning på svenska ix Introduction 1 Post-translational modifications 1 Nucleotidylylation and phosphocholination 2 Small GTPases 7 Pathogens modify host cells at a molecular level 9 Quorum sensing in Legionella pneumophila 12 Proteomics towards PTMs 13 Chapter 1: Towards the identification of adenylylated proteins and adenylylation-modifying enzymes (Paper I – III) 19 Previous work 19 Outline: From building blocks to antibodies 22 Synthesis of a tyrosine-AMP building block 23 Synthesis of a threonine- and serine-AMP building block 26 Synthesis of adenylylated Peptides 28 Generation of AMP specific antibodies 30 Mass fragmentation patterns of adenylylated peptides 37 Immunoprecipitation of adenylylated proteins 42 Non-hydrolysable mimics for the study of deadenylylating enzymes 47 Future work 52 Ongoing Work – Covalent trapping of substrates of adenylyl transferases 53 Conclusions 60 Chapter 2: Tools for
    [Show full text]
  • Communication © 1998 by the American Society for Biochemistry and Molecular Biology, Inc
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 12, Issue of March 20, pp. 6595–6598, 1998 Communication © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Second Mammalian 2). Most myristoylated proteins are acylated through an amide linkage to their N-terminal glycine residues, a reaction cata- N-Myristoyltransferase* lyzed by the enzyme, N-myristoyltransferase (NMT)1 (EC 2.3.1.97) (3–5). Myristoylation has proven essential to the bio- (Received for publication, December 23, 1997, and in revised logical activity of many mammalian, viral, and fungal proteins. form, January 21, 1998) In particular, the transformation potential of the protein tyro- Dan K. Giang and Benjamin F. Cravatt‡ sine kinase, p60src, is entirely dependent on myristoylation, as src From the Skaggs Institute for Chemical Biology and nonmyristoylated forms of p60 fail to bind cellular mem- Department of Cell Biology, The Scripps Research branes and are transformation defective (6). Similarly, non- Institute, La Jolla, California 92037 myristoylated forms of endothelial nitric-oxide synthase are not properly localized to the Golgi apparatus and plasma- N-terminal myristoylation is a cotranslational lipid modification common to many signaling proteins that lemmal caveolae, resulting in marked reductions in stimulated often serves an integral role in the targeting and/or nitric oxide production (7, 8). The dependence of viral infectiv- function of these proteins. Myristoylation is catalyzed ity on myristoylation is exemplified by the observation that by an enzyme activity, N-myristoyltransferase (NMT), inhibiting the myristoylation of the human immunodeficiency which transfers myristic acid from myristoyl coenzyme virus type I GAG precursor protein promotes the production of A to the amino group of a protein’s N-terminal glycine noninfectious viral particles (9).
    [Show full text]
  • The Functional Significance of G Protein -Coupled Receptor Dimerization
    THE FUNCTIONAL SIGNIFICANCE OF G PROTEIN-COUPLED RECEPTOR DIMERIZATION by Matthew R. Whorton A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2008 Doctoral Committee: Assistant Professor Roger K. Sunahara, Chair Assistant Professor Jorge A. Iñiguez-Lluhí Professor Jennifer J. Linderman Professor Richard R. Neubig Professor William B. Pratt Associate Professor John J. G. Tesmer To my parents and Jen for their love, support, and encouragement. ii ACKNOWLEDGEMENTS I would like to thank my advisor Roger Sunahara for his guidance and support. I would also like to extend my gratitude to other members of the Sunahara lab, both past and present, as well as members of the Neubig and Tesmer labs for thoughtful discussions and comments during lab meetings. I would also like to extend a great deal of gratitude to several collaborators who have provided their expertise to various aspects of my research. At Stanford University, Mike Bokoch and Soren Rasmussen in Brian Kobilka’s lab and Bo Huang in Richard Zare’s lab were instrumental in fluorescently labeling β2AR and then imaging it with their powerful TIRF single molecule setup. Beata Jastrzebska and Paul Park in Krzysztof Palczewski’s lab at Case Western University were also invaluable in providing purified rhodopsin as well as performing transducin activation assays. And finally, I would like to thank Dimitrios Fotiadis in Andrea Engel’s lab for using their various microscopy techniques to image rhodopsin-HDL complexes. I would also like to thank my friends and roommates through out the years who have helped to enrich the graduate school experience.
    [Show full text]
  • And Post-Translationally N-Myristoylated Proteomes in Human Cells
    ARTICLE Received 5 Mar 2014 | Accepted 5 Aug 2014 | Published 26 Sep 2014 DOI: 10.1038/ncomms5919 OPEN Global profiling of co- and post-translationally N-myristoylated proteomes in human cells Emmanuelle Thinon1,2,*,w, Remigiusz A. Serwa1,*, Malgorzata Broncel1, James A. Brannigan3, Ute Brassat1,2,w, Megan H. Wright1,4,w, William P. Heal1,w, Anthony J. Wilkinson3, David J. Mann2,4 & Edward W. Tate1,4 Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of 4100 N-myristoylated proteins, 495% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for 470 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. 1 Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, UK. 2 Department of Life Sciences, Imperial College London, Exhibition Road, London SW7 2AZ, UK. 3 York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.
    [Show full text]
  • A Bioorthogonal Chemical Reporter for Fatty Acid Synthase-Dependent Protein Acylation 2 3 Krithika P
    bioRxiv preprint doi: https://doi.org/10.1101/2021.05.07.443132; this version posted May 7, 2021. 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. 1 A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation 2 3 Krithika P. Karthigeyan1, Lizhi Zhang2, David R. Loiselle3, Timothy A. J. Haystead3, 4 Menakshi Bhat1, Jacob S. Yount2*, Jesse J. Kwiek1* 5 1. Department of Microbiology and Center for Retrovirus Research, The Ohio State 6 University, Columbus, Ohio, USA. 7 2. Department of Microbial Infection and Immunity, The Ohio State University, Columbus, 8 Ohio, USA. 9 3. Department of Pharmacology and Cancer Biology, Duke University School of Medicine, 10 Durham, NC, USA. 11 *Co-corresponding authors. Kwiek.2@osu.edu, Jacob.Yount@osumc.edu 12 13 Summary 14 Cells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid 15 synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers 16 and during replication of many viruses. The precise role of FASN in disease pathogenesis is 17 poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein 18 acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry 19 compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN- 20 dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the 21 cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, 22 and the HIV-1 matrix protein at its myristoylation site.
    [Show full text]
  • ( 12 ) United States Patent ( 10 ) Patent No .: US 10,882,885 B2 Wallace Et Al
    US010882885B2 ( 12 ) United States Patent ( 10 ) Patent No .: US 10,882,885 B2 Wallace et al . ( 45 ) Date of Patent : Jan. 5 , 2021 ( 54 ) PEPTIDE - BASED SYNTHETIC MOLECULES OTHER PUBLICATIONS AND SILICA NANOSTRUCTURES Lechner et al. ( “ Exploring the effect of native and artificial peptide modifications on silaffin induced silica precipitation, ” Chem . Sci . , ( 71 ) Applicant: Massachusetts Institute of 2012 , 3 , 3500 ) ( Year: 2012 ) . * Technology , Cambridge, MA (US ) Seker et al . “ Engineered Peptides for Nanohybrid Assemblies , " Langmuir 2014 , 30 , 2137-2143 ( Year: 2014 ) . * ( 72 ) Inventors: Andrea Kimi Wallace, Cambridge , MA Pender et al . “ Peptide - Mediated Formation of Single -Wall Carbon ( US ) ; Maiko Furubayashi , Cambridge , Nanotube Composites , ” Nano Letters 2006 , 6 , 40-44 ( Year: 2006 ) . * Coyle et al . “ A Cleavable Silica -Binding Affinity Tag for Rapid and MA ( US ) ; Christopher A. Voigt, Inexpensive Protein Purification ,” Biotechnol . Bioeng . 2014 ; 111 : Belmont, MA (US ) 2019-2026 ( Year: 2014 ) . * Nguyen et al. A protein -protein interaction in magnetosomes : TPR ( 73 ) Assignee : Massachusetts Institute of protein MamA interacts with an Mms6 protein , Biochem Biophys Technology , Cambridge, MA (US ) Rep . 2016 ; 7 : 39-44 ( Year: 2016 ) . * Uniprot entry for concentrative nucleoside transporter 1 , down loaded May 5 , 2020 ( Year: 2020 ) . * ( * ) Notice : Subject to any disclaimer , the term of this Uniprot entry for sodium / nucleoside contransporter , downloaded patent is extended or adjusted under 35 May 5 , 2020 ( Year: 2020 ) . * U.S.C. 154 ( b ) by 0 days. [ No Author Listed ] , THAOC_24272 . UniProt . Nov. 28 , 2012. 5 pages . Retrieved from : http://www.uniprot.org/uniprot/KORQ67. [ No Author Listed ] , THAOC_37500 . UniProt . Nov. 28 , 2012. 5 ( 21 ) Appl. No .: 15 /897,257 pages.
    [Show full text]