European School of Molecular Medicine (SEMM) Naples site Università degli Studi di Napoli “Federico II” PhD in Molecular Medicine –VI/XXIV cycle Human Genetics Unravelling aldolase C moonlighting functions: a multi-task approach PhD candidate: Simona Langellotti Tutor: Prof. Francesco Salvatore Internal Supervisor: Prof. Stefania Orrù External Supervisor: Prof. Francisco Ernesto Baralle Academic Year 2011/2012 TABLE OF CONTENTS LIST OF ABBREVIATIONS ...............................................................................................3 FIGURE INDEX ...................................................................................................................4 TABLES INDEX ...................................................................................................................5 ABSTRACT ..........................................................................................................................6 SPECIFIC AIMS ..................................................................................................................9 Chapter 1 ............................................................................................................................10 INTRODUCTION ...............................................................................................................10 1.1 The aldolase system..........................................................................................10 1.2 Aldolase Isozymes. ............................................................................................11 1.3 Protein structure. ................................................................................................15 1.4 The aldolase C isozyme....................................................................................17 1.5 Aldolase C gene expression during development.........................................19 1.6 Aldolase “moonlighting” functions and involvement of aldolase C in the physiology of the CNS.......................................................................................20 1.7 Aldolase C and the NF-L mRNA: a possible involvement in neurodegeneration. ............................................................................................24 1.8 Proteomics and the functional proteomic approach. ....................................27 1.9 Monoclonal antibodies: features and production...........................................30 Chapter 2 ............................................................................................................................34 MATERIALS AND METHODS ........................................................................................34 2.1 Standard solutions. ............................................................................................34 2.2 Enzymes. .............................................................................................................34 2.3 Synthetic oligonucleotides. ...............................................................................35 2.4 Radioactive isotope............................................................................................35 2.5 Bacterial culture..................................................................................................35 2.6 Cell culture...........................................................................................................35 2.7 DNA preparation.................................................................................................36 2.7.1 Small scale preparation of plasmid DNA from bacterial cultures. ..............36 2.7.2 Large scale preparations of plasmid DNA from bacterial cultures. ............37 2.8 RNA extraction from cultured cells. .................................................................38 2.9 Estimation of nucleic acid concentration. .......................................................38 2.10 cDNA synthesis. .................................................................................................39 2.11 PCR amplification of selected DNA fragments..............................................39 2.12 Enzymatic modification of DNA........................................................................44 2.12.1 Restriction enzymes...........................................................................................44 2.12.2 T4 DNA ligase.....................................................................................................44 2.13 Agarose gel electrophoresis of DNA...............................................................45 2.14 Elution and purification of DNA fragments from agarose gels. ...................45 2.15 Preparation of bacterial competent cells. .......................................................46 2.16 Transformation of bacteria................................................................................46 2.17 Cell culture transfections...................................................................................47 2.18 Anti-FLAG immunoprecipitation (IP) and protein(s) co-immunoprecipitation (co-IP)...................................................................................................................47 2.19 SDS-PAGE and In situ hydrolysis of proteins................................................48 2.20 Protein identification by mass spectrometry and bioinformatics.................49 2.21 Protein extraction from cells or tissues...........................................................50 2.22 Western blotting (immunoblotting)...................................................................51 2.23 In vitro transcription............................................................................................52 2.24 Protein-RNA co-immunoprecipitation assay. .................................................54 2.25 Real Time PCR...................................................................................................55 2.26 Expression and purification of fusion proteins...............................................56 1 2.27 RNA EMSA..........................................................................................................57 2.28 UV cross-linking experiments...........................................................................58 2.29 Elisa assay of biotinylated peptides. ...............................................................59 2.30 Aldolase C immunoprecipitation using the 9F mAb......................................61 Chapter 3 ............................................................................................................................62 RESULTS AND DISCUSSION........................................................................................62 PART A...............................................................................................................................62 A functional proteomics approach to identify novel molecular interactors of the aldolase C protein: from a new binding partner of aldolase C to the study of its putative RNA-binding ability. ...........................................................................................62 3A.1 Immunoprecipitation of 3xFLAG-tagged aldolase C protein in Neuro2a cells.......................................................................................................................62 3A.2 Mass spectrometry identification of aldolase C interacting proteins. .........66 3A.3 Validation of the interaction between aldolase C and the 14-3-3 γ protein by Western Blotting............................................................................................72 3A.4 The NF-L mRNA: a possible functional link between aldolase C and the 14-3-3 γ protein...................................................................................................73 3A.4.1 The NF-L RNA co-immunoprecipitates with aldolase C...............................75 3A.4.2 EMSA and supershift tests confirm the presence of aldolase C within an NF-L mRNA-binding complex but demonstrate that no direct binding occurs between aldolase C and this RNA target...........................................77 3A.4.3 The protein-RNA UV cross-linking assay confirm the absence of direct binding between aldolase C and the NF-L mRNA. .......................................82 3A.5 Evaluation of aldolase C expression response after NGF administration in the PC12 cell line. ..............................................................................................84 PART B …………………………………………………………………………………...88 Antigenic characterization of monoclonal anti-human aldolase C antibodies. ........88 3B.1 Monoclonal antibodies preparation. ................................................................89 3B.2 Isozyme specificity of the 9F and 2A mAbs. ..................................................89 3B.3 Identification of the aldolase C protein “macrodomain” including the specific antigen recognized by the 9F and 2A mAbs. ..................................91 3B.4 Aldolase A vs aldolase C: sequence alignment of residues 1-100. ...........95 3B.5 “ElliPro” analysis of the tertiary structure of the human aldolase C protein. ...............................................................................................................................96 3B.6 ELISA screening of biotinylated aldolase C peptides.................................102 3B.7 Epitope mapping in eukaryotic cells confirms specificity of the 9F and 2A mAbs for peptide 85-102.................................................................................105