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Protein Prenylation Reactions As Tools for Site-Specific Protein Labeling and Identification of Prenylation Substrates

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Protein Prenylation Reactions As Tools for Site-Specific Protein Labeling and Identification of Prenylation Substrates Protein prenylation reactions as tools for site-specific protein labeling and identification of prenylation substrates Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereichs Chemie der Technischen Universität Dortmund Angefertigt am Max-Planck-Institut für molekulare Physiologie in Dortmund Vorgelegt von Dipl.-Chemikerin Thi Thanh Uyen Nguyen aus Jülich Dortmund, April 2009 Die vorliegende Arbeit wurde in der Zeit von Oktober 2005 bis April 2009 am Max-Planck- Institut für molekulare Physiologie in Dortmund unter der Anleitung von Prof. Dr. Roger S. Goody, Prof. Dr. Kirill Alexandrov und Prof. Dr. Herbert Waldmann durchgeführt. 1. Gutachter : Prof. Dr. R. S. Goody 2. Gutachter : Prof. Dr. H. Waldmann The results of this work were published in the following journals: “Exploiting the substrate tolerance of farnesyltransferase for site-selective protein derivatization” U.T.T. Nguyen, J. Cramer, J. Gomis, R. Reents, M. Gutierrez-Rodriguez, R.S. Goody, K. Alexandrov, H. Waldmann, Chembiochem 2007, 8 (4), 408-23. “Development of selective RabGGTase inhibitors and crystal structure of a RabGGTase- inhibitor complex” Z. Guo, Y.W. Wu, K.T. Tan, R.S. Bon, E. Guiu-Rozas, C. Delon, U.T.T. Nguyen, S. Wetzel,S. Arndt, R.S. Goody, W. Blankenfeldt, K. Alexandrov, H. Waldmann, Angew Chem Int Ed Engl 2008, 47 (20), 3747-50. “Analysis of the eukaryotic prenylome by isoprenoid affinity tagging” U.T.T. Nguyen, Z. Guo, C. Delon, Y.W. Wu, C. Deraeve, B. Franzel, R.S. Bon, W. Blankenfeldt, R.S. Goody, H. Waldmann, D. Wolters, K. Alexandrov, Nat Chem Biol 2009, 5 (4), 227-35. Ba Má Contents................……………………………………………………………………………………..i Abbreviations............………………………………………………………………………………….iii 1 INTRODUCTION _____________________________________________________ 1 1.1 Posttranslational modifications ______________________________________1 1.2 Protein prenylation_________________________________________________2 1.2.1 Structural features of the protein prenyltransferases ____________________________ 3 1.2.2 Mechanism of the protein prenylation reaction _________________________________ 8 1.3 Protein substrates of the protein prenyltransferases____________________10 1.3.1 The small GTPases of the Ras superfamily __________________________________ 10 1.3.2 Ras and Rho/Rac/Cdc42 families: post-prenylation processing, localization, and signaling _____________________________________________________________ 11 1.3.3 The vesicular transport controlled by RabGTPases ____________________________ 16 1.3.4 Protein Prenyltransferases as therapeutic targets _____________________________ 22 1.4 Tools to investigate protein prenylation ______________________________24 1.5 Strategies for site-specific protein labeling____________________________28 2 AIMS OF THE PROJECT _____________________________________________ 35 3 RESULTS AND DISCUSSION _________________________________________ 39 3.1 Exploiting the substrate tolerance of FTase for site-specific protein labeling39 3.1.1 Strategy for the design of the genetically encodable microtag and the chemically functionalized isoprenoid analogs__________________________________________ 39 3.1.2 Prior work ____________________________________________________________ 41 3.1.3 The isoprenoid analogs as efficient lipid donors for the prenylation reaction catalyzed by FTase _______________________________________________________________ 43 3.1.4 Biochemical features of proteins derivatized with the isoprenoid analogs ___________ 49 3.1.5 Further chemoselective derivatization of the prenylated proteins by Staudinger ligation and Diels-Alder cycloaddition _____________________________________________ 53 3.2 Towards the isolation and analysis of the cellular mammalian prenylome __59 3.2.1 BGPP as an efficient lipid donor for the RabGGTase-mediated prenylation reaction in vitro_________________________________________________________________ 60 3.2.2 BGPP as an efficient lipid donor for the RabGGTase-mediated prenylation of endogenous RabGTPases in COS-7 lysate ____________________________________________ 62 3.2.3 Enrichment of biotin-geranylated RabGTPases by means of streptavidin chromatography _______________________________________________________ 64 3.2.4 Engineering FTase and GGTase-I mutants capable of BGPP transfer _____________ 67 3.2.5 Analysis of the entire cellular prenylome ____________________________________ 78 3.2.6 Effect of protein prenyltransferase inhibitors on the prenylome ___________________ 78 i 3.2.7 Quantitative analysis of the mammalian prenylome ___________________________ 83 4 SUMMARY AND OUTLOOK ___________________________________________89 5 MATERIALS AND METHODS __________________________________________95 5.1 Materials ________________________________________________________ 95 5.1.1 General Materials _____________________________________________________ 95 5.1.2 Other chemicals from contributing people ___________________________________ 96 5.1.3 Plasmids ____________________________________________________________ 96 5.1.4 General instrumentation ________________________________________________ 97 5.1.5 Frequently used buffers and growth media __________________________________ 98 5.2 Protein expression and purification methods _________________________ 99 5.2.1 Expression and purification of CFP-CAAX __________________________________ 99 5.2.2 Expression and purification of FTasewt, GGTase-Iwt and their respective mutants, RabGGTasewt, REP-1, MRS6p, and the small GTPases _______________________ 99 15 5.2.3 Expression and purification of [ N]-labelled Rab22A __________________________ 99 5.3 Analytical methods _______________________________________________ 99 5.3.1 LC-ESI-MS __________________________________________________________ 99 5.3.2 MALDI-TOF mass spectrometry _________________________________________ 100 5.3.3 Gel filtration chromatography (GF) _______________________________________ 100 5.3.4 Denaturing SDS-PAGE (1D and 2D) ______________________________________ 101 5.3.5 Western blot analysis__________________________________________________ 101 5.4 Biochemical methods ____________________________________________ 102 5.4.1 In vitro protein prenylation ______________________________________________ 102 5.4.2 Streptavidin pulldown__________________________________________________ 103 5.4.3 In lysate protein prenylation_____________________________________________ 103 5.4.4 Quantification of RabGTPase abundance in COS-7 lysate _____________________ 104 5.4.5 Chemoselective modification of the prenylated proteins _______________________ 104 5.5 Biophysical methods_____________________________________________ 105 5.5.1 Fluorescence titrations- determination of KD ________________________________ 105 5.5.2 Continuous fluorometric assay for FTase and GGTase-I prenylation _____________ 106 5.6 COS-7 cells general maintenance and lysate preparation_______________ 107 5.7 Crystallization and structure solution of the BGPP:FTase and the BGPP: FTaseW102T_Y154T complexes ________________________________________ 107 6 REFERENCES _____________________________________________________109 7 ACKNOWLEDGEMENTS _____________________________________________131 8 DECLARATION/EIDESSTATTLICHE ERKLÄRUNG _______________________133 ii ABBREVIATIONS A Ampère Å Angstrom (1 Å = 0.1 nm = 10-10 m) AA Amino acid ACN Acetonitrile BG Biotin-geranyl BGPP Biotin-geranyl pyrophosphate CBR C-terminal binding region CHM Choroideremia CIM CBR interacting motif COMP compactin Da Dalton Dans / Dansyl 5-Dimethylaminonaphtalin-1-sulfonyl DMSO Dimethylsulfoxide DTE 1,4-Dithioerythritol DTT 1,4-Dithiothreitol F Farnesyl FPP Farnesylpyrophosphate FTase Farnesyltransferase GAP GTPase activating protein GDF GDI displacement factor GDI GDP dissociation inhibitor GdmHCl Guanidinium hydrochloride GEF Guaninenucleotide exchange factor GF Gel filtration GG Geranylgeranyl GGPP Geranylgeranylpyrophosphate GGTase-I Geranylgeranyltransferase-I GGTI GGTase-I inhibitor GST Glutathione S-transferase GTPase Guaninetriphosphate phosphatase h hour HOPS Homotypic fusion and vacuole protein sorting HPLC High performance liquid chromatography Icmt Isoprenylcysteine carboxymethyltransferase IG Immunoglobulin IPTG Isopropyl-β-D-thiogalactoside iii LC-MS Liquid chromatography-mass spectrometry LRR Leucine-rich repeat MALDI-TOF-MS Matrix assisted laser desorption/ionization-time of flight mass spectrometry mant N-methylanthraniloyl min minute MudPIT Multidimensional Protein Identification Technology MWCO Molecular weight cut off NBD 7-Nitrobenz-2-oxa-1,3-diazol-4-yl NSF N-ethyl-maleimide sensitive factor OD600 Optical density at 600 nm PP Pyrophosphate PTM Posttranslational modification Rab Ras-like (protein) from Rat brain RabGGTase Rab Geranylgeranyltransferase Ras Rat adeno sarcoma REP Rab escort protein Rho Ras homologous protein RP-HPLC Reversed-phase high performance liquid chromatography RT Room temperature SDS Sodium dodecyl sulfate Sec Secretory protein SNAP Soluble NSF association protein SNARE Soluble NSF attachment protein receptor TFA Trifluoroacetic acid wt wild-type Ypt Yeast protein transport iv INTRODUCTION 1 INTRODUCTION 1.1 Posttranslational modifications The deciphering of the human genome in 2001 constitutes a milestone in the history of biological research. Unexpectedly, it revealed that it contains just 30.000 genes; this is only twice as many as in lower eukaryotes such as worm or fly1, 2. Since the size of the human proteome, defined as the number of different
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