University of Pisa
DEPARTMENT OF BIOLOGY
DOCTORATE COURSE IN MOLECULAR BIOTECHNOLOGY
BIO
MOLECULAR
S
CIENCE,
BIOS
SCHOOL
XXIV CYCLE
“Structural and functional studies of cytosolic
5’-nucleotidase II (cN-II)”
(SSD BIO/10)
Tutor:
Prof. Maria Grazia Tozzi
Candidate:
Dr. Daniela Nicole Filoni
The good samaritan (1867), Rodolphe Bresdin, lithograph The New York Public Library, Astor, Lenox and Tilden Foundations
Grazie a Via San Zeno 51, perchè ha segnato la nascita della Academy of Sfigology. Grazie a Via San Zeno 51, perché ha dato un nuovo sapore ai tartufi neri, anche agli scorzoni. Grazie a Via San Zeno 51, perché ha narrato con altra voce
Alice in wonderland, che adesso – la mia Alice – danza nei meandri di Metaboland. Grazie a Via San Zeno 51, perché ha dipinto di nuovi colori il mio mondo. Grazie a Via San Zeno 51, perché mi ha regalato un caleidoscopio di emozioni.
Grazie a Via San Zeno 51, perché ha reso le mie mani portatrici di significato. Grazie a Via San Zeno 51, perché appena vi entro è una
gustosa satura lanx. Grazie a Via San Zeno 51, perché adesso risuona in me non solo il mio eco. Ma soprattutto grazie a Gabriele,
che mi aiuta tutti i giorni ad esplorare ogni angolo di noi.
INDEX
ABSTRACT 1
INTRODUCTION 3
1. The mammalian 5’-Nucleotidases 3
1.1 Ecto 5’-Nucleotidase 4
1.2 Intracellular Nucleotidases and HAD superfamily 12 1.2.1 Cytosolic 5’-Nucleotidases IA and IB 15 1.2.2 Cytosolic 5’-Nucleotidase III 19 1.2.3 Cytosolic 5’ (3’)-deoxyribonucleotidase 25 1.2.4 Mitochondrial 5’(3’)-deoxyribonucleotidase 27
1.3 Cytosolic 5’-Nucleotidase II 33
1.3.1 Phylogenetic, tissue and subcellular distribution 33
1.3.2 Reaction mechanism 35 1.3.3 Regulation 39 1.3.4 Structure 43 1.3.5 Physiological role 53 1.3.6 Terapeutical interest 59 1.3.6.1 Lesch-Nyhan syndrome 59
1.3.6.2 CN-II and prodrugs 60
2. Aim of the project 66
MATERIALS AND METHODS 67
1. Materials 67
2. Methods 68
2.1 Molecular biology methods 68
2.1.1 PET expression system and site-directed mutagenesis 68 2.1.2 Preparation and transformation of competent cells 72 2.1.3 Cloning of shRNA into conditional LV 73
2.2 Biochemistry methods 80
2.2.1 Expression and purification of the recombinant proteins 80 2.2.2 Preparation of cell crude extract 81
2.2.3 cN-II assays 81
2.2.3.2 Phosphotransferase activity 82 2.2.3.3 Free phosphate colorimetric detection 82 2.2.3 Western and immunoblotting 83 2.2.4 Gel filtration chromatography 84 2.2.5 Discontinuos native gradient electrophoresis 85
2.3 Cell biology methods 86
2.3.1 Cell culture 86
2.3.2 Lentivirus production 87
2.3.3 Cell transduction 88
2.3.4 MTT assay 89
2.4 Methods in collaboration 90
RESULTS AND DISCUSSION 91
1. Structural and Regulatory features 91
1.1 Biochemical characterization of point mutants 91
1.1.1 Effector sites 91
1.1.2 Interfaces 97
1.2 Structural studies of WT and mutated recombinant cN-II 100
2. Physiological aspects 105
2.1 CN-II expression in Saccharomyces cerevisiae 105 2.2 CN-II silencing in human cell lines 107
CONCLUSIONS 117
1. Structural and regulatory features 117
2. Physiological aspects 119
ABBREVIATIONS 122
BIBLIOGRAPHY 126
