POST-TRANSLATIONAL MODIFICATIONS BY SUMO AND UBIQUITIN-LIKE PROTEINS; ROLE OF SUMOYLATION ON SALL PROTEINS Lucia Pirone January 2016 (cc)2016 LUCIA PIRONE (cc by-nc 4.0) Facultad de Ciencia y Tecnología Departamento de Genética, Antropología Física y Fisiología Animal POST-TRANSLATIONAL MODIFICATIONS BY SUMO AND UBIQUITIN-LIKE PROTEINS; ROLE OF SUMOYLATION ON SALL PROTEINS Memoria presentada por Lucia Pirone Enero 2016 Este trabajo ha sido realizado en la unidad de Genómica Funcional del Centro de Investigación Cooperativa en Biociencias (CIC bioGUNE), financiado con una beca doctoral del proyecto UPStream: European Research Training in the Ubiquitin Proteasome System (Marie Curie Initial Training Network, ITN, FP7A-PEOPLE 2011-ITN) bajo la dirección de la Doctora María Rosa Barrio Olano INDEX ABBREVIATIONS ............................................................................................................................ 1 FIGURES & TABLES ....................................................................................................................... 5 SUMMARY ........................................................................................................................................ 9 RESUMEN ........................................................................................................................................ 10 I. INTRODUCTION ........................................................................................................................ 11 1. Post-translational modifications ................................................................................................. 13 1.1. SUMOylation and SUMO proteins .................................................................................... 14 1.2. The SUMOylation pathway ............................................................................................... 14 1.3. SUMOylation consensus motifs versus SUMO interaction ............................................... 17 1.3.1. SUMO consensus motif .................................................................................................. 18 1.3.2. SUMO non-covalent interactions ................................................................................... 18 1.4. Strategies to study SUMO modification ............................................................................ 19 1.5. The SUMOylation process in Drosophila ......................................................................... 20 1.5.1. Drosophila melanogaster as a model system ................................................................. 20 1.5.2. SUMOylation pathway in Drosophila ............................................................................ 22 1.5.3. The role of SUMOylation in Drosophila ....................................................................... 22 1.6. UFM1 and the UFMylation pathway ................................................................................. 23 2. The SALL family of transcription factors ................................................................................. 24 2.1. SALL proteins in human health ......................................................................................... 27 2.2. Post-translational modification of SALL proteins ............................................................. 28 2.3. Role of SALL proteins during wing development in Drosophila ..................................... 29 2.3.1. Development of the wing imaginal disc ......................................................................... 30 2.3.2. SALL function during wing development ...................................................................... 31 II. OBJECTIVES ............................................................................................................................. 33 III. HYPOTHESIS ........................................................................................................................... 37 IV. MATERIALS & METHODS ................................................................................................... 41 1. Generation of vectors ................................................................................................................... 43 2. Cell culture.................................................................................................................................... 46 2.1. Cell transfections................................................................................................................ 46 2.2. NeutrAvidin pulldown ....................................................................................................... 48 2.3. GFP-Trap co-pulldown ...................................................................................................... 48 2.4. Immunofluorescence in cultured cells ............................................................................... 49 3. Western blot .................................................................................................................................. 51 4. In vitro SUMOylation and SUBEs pulldown ............................................................................. 52 5. Proximity Ligation Assays ........................................................................................................... 53 6. Mass Spectrometry ....................................................................................................................... 53 7. Drosophila husbandry and stocks ............................................................................................... 54 7.1. Generation of transgenic flies ............................................................................................ 57 7.2. Isolation of bioSmt3 conjugates in vivo ............................................................................. 57 7.3. Analysis of adult wing phenotype ...................................................................................... 58 7.4. Immunohistochemistry ....................................................................................................... 60 8. Statistical analysis ........................................................................................................................ 60 V. RESULTS ..................................................................................................................................... 61 PART I: ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS BY UBIQUITIN- LIKES ................................................................................................................................................ 63 1. Identification of SUMOylated proteins in Drosophila melanogaster. ...................................... 63 1.1. New methodology for the isolation of SUMOylated proteins in Drosophila cells ............ 63 1.2. Localization of bioSmt3 in Drosophila cells ..................................................................... 66 1.3. SUMOylation of Ftz-f1 by bioSmt3................................................................................... 67 1.4. Isolation and identification of new bioSmt3 conjugates in Drosophila cultured cells ...... 69 1.5. Validation of bioSmt3 conjugates in Drosophila cultured cells............................................. 72 1.6. Isolation of bioSmt3 conjugates in vivo ............................................................................. 74 1.7. Localization of bioSmt3 conjugates in vivo ....................................................................... 76 1.8. Identification of bioSmt3 conjugates in vivo ...................................................................... 77 2. Development of bioUbiquitin-like (bioUbL) vectors for the analysis of post-translational modifications in mammalian cells ................................................................................................... 81 2.1. New methodology for the isolation of SUMOylated proteins in mammalian cells ........... 81 2.2. Isolation and identification of SUMO3 subproteome ........................................................ 81 2.3. New methodology for the isolation of proteins modified by other UbLs in mammalian cells ............................................................................................................................................ 83 2.4. Validation of the bioUFM1 system using a known target ................................................. 85 2.5. Isolation and identification of UFM1 conjugates ............................................................... 86 PART II: ANALYSIS OF THE SUMOYLATION OF SALL PROTEINS ............................... 89 1. SUMOylation and localization of human SALL1 ..................................................................... 89 1.1. Human SALL1 is SUMOylated in cultured cells............................................................... 89 1.2. SALL1 localizes in nuclear bodies in human cells ............................................................ 91 1.2.1. Human SALL1 partially colocalizes with SUMO proteins ............................................ 92 1.2.2. Human SALL1 interacts with SUMO proteins by PLA ................................................. 94 2. SUMOylation and localization of Drosophila Sall Proteins ..................................................... 96 2.1. SUMOylation of Drosophila Salm in mammalian cells .................................................... 97 2.2. Drosophila Salm localizes in nuclear bodies in cultured cells .........................................