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Characterization of the Role of USP25 in EGFR Endocytosis

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Characterization of the Role of USP25 in EGFR Endocytosis PhD degree in Molecular Medicine (curriculum in Molecular Oncology) European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Settore disciplinare: Bio/10 Characterization of the role of USP25 in EGFR endocytosis Nadine Caroline Woessner IFOM, Milan Matricola n. R08892 Supervisor: Dr. Simona Polo IFOM, Milan Anno accademico 2013-2014 TABLE OF CONTENTS LIST OF ABBREVIATIONS................................................................................................ 8 FIGURE AND TABLE INDEX .......................................................................................... 11 ABSTRACT ........................................................................................................................ 13 INTRODUCTION ............................................................................................................... 15 1 Endocytosis .................................................................................................................. 15 1.1 Endocytic entry routes .......................................................................................... 15 1.1.1 Clathrin-mediated endocytosis (CME) .......................................................... 16 1.1.2 Non-clathrin endocytosis (NCE) ................................................................... 16 1.2 Endocytic sorting .................................................................................................. 19 1.3 Transferrin as a model substrate for CME ............................................................ 20 1.4 Endocytosis and signalling.................................................................................... 21 1.4.1 Endocytosis regulates signalling.................................................................... 21 1.4.2 Signalling regulates endocytosis.................................................................... 23 1.4.3 Signalling endosomes .................................................................................... 25 2 The ubiquitin system .................................................................................................... 26 2.1 Ubiquitin in endocytosis ....................................................................................... 29 2.1.1 Ubiquitination of cargoes .............................................................................. 30 2.1.2 Ubiquitin and endocytic adaptors .................................................................. 31 2.1.3 Ubiquitin in endosomal sorting ..................................................................... 33 2.2 Deubiquitinating enzymes (DUBs) in endocytosis ............................................... 35 2.2.1 Mechanism of catalysis .................................................................................. 36 2 2.2.2 DUB families .................................................................................................37 2.2.3 DUB activity in early steps of endocytosis ....................................................39 2.2.4 DUB activity in later steps of endocytosis .....................................................40 2.2.5 DUB activity in the secretory pathway ..........................................................43 3 The EGFR system ........................................................................................................44 3.1 Ligand-induced EGFR signal transduction ...........................................................45 3.2 EGFR endocytosis and trafficking ........................................................................46 3.3 Role of ubiquitin in EGFR endocytosis ................................................................47 3.4 Role of DUBs in EGFR endocytosis .....................................................................51 4 USP25 ..........................................................................................................................52 5 Cullin 3 .........................................................................................................................55 5.1 Structure and regulation of Cullins........................................................................55 5.2 Cullin 3 in endocytosis ..........................................................................................56 MATERIAL AND METHODS ...........................................................................................58 1 Solutions .......................................................................................................................58 1.1 Phosphate-buffered saline .....................................................................................58 1.2 Tris-HCl (1 M) ......................................................................................................58 1.3 Tris-buffered saline (TBS) ....................................................................................58 1.4 10X SDS-PAGE running buffer ............................................................................59 1.5 10X transfer buffer ................................................................................................59 1.6 50X TAE (Tris-Acetate-EDTA) ............................................................................59 2 Protein buffers ..............................................................................................................59 3 2.1 1X JS buffer .......................................................................................................... 59 2.2 1X RIPA buffer ..................................................................................................... 60 2.3 1X Laemmli buffer................................................................................................ 60 3 Reagents ....................................................................................................................... 61 3.1 Antibodies ............................................................................................................. 61 3.2 RNAi oligos .......................................................................................................... 62 3.2.1 Negative control siRNA ................................................................................ 62 3.2.2 Specific RNAi oligos ..................................................................................... 62 3.3 TaqMAN assays for qRT-PCR (Applied Biosystems) ......................................... 62 4 Cloning techniques ...................................................................................................... 63 4.1 Agarose gel electrophoresis .................................................................................. 63 4.2 Minipreps .............................................................................................................. 63 4.3 Diagnostic DNA restriction .................................................................................. 63 4.4 Large scale plasmid preparation ........................................................................... 63 4.5 Transformation of competent cells ....................................................................... 64 5 Constructs and plasmids .............................................................................................. 64 6 Cell culture ................................................................................................................... 65 6.1 Cell culture media ................................................................................................. 65 6.2 Transfections ......................................................................................................... 65 6.2.1 RNAi transfections ........................................................................................ 65 6.2.2 DNA transfections ......................................................................................... 66 6.3 Retroviral and lentiviral infection ......................................................................... 66 4 7 Protein procedures ........................................................................................................67 7.1 Cell lysis ................................................................................................................67 7.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) .......................................67 7.3 Immunoblot (IB) ....................................................................................................68 7.4 Anti-ubiquitin immunoblot ....................................................................................69 7.5 Immmunoprecipitation ..........................................................................................69 8 DUB library screening .................................................................................................70 9 Protein production and purification .............................................................................70 9.1 GST-fusion protein production .............................................................................70 9.2 GST pull-down ......................................................................................................72 10 Assays with 125I-EGF and 125I-Tf .................................................................................72 10.1 Receptor internalization assays with 125I-EGF and 125I-Tf ................................72 10.2 Measurement of the number of EGF/Tf receptors at the cell surface by saturation binding
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