UNIVERSITأ€ DEGLI STUDI DI 6.3 Aggregation of as variants in the presence of DHA 102 7....

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  • UNIVERSITÀ DEGLI STUDI DI PADOVA

    Dipartimento di Biologia

    Centro di Ricerca Interdipartimentale per le Biotecnologie Innovative

    - CRIBI -

    SCUOLA DI DOTTORATO DI RICERCA IN BIOSCIENZE E BIOTECNOLOGIE

    INDIRIZZO DI BIOTECNOLOGIE

    XXVI CICLO

    α -SYNUCLEIN OLIGOMERS INDUCED BY DOCOSAHEXAENOIC ACID

    A STUDY OF ACTIVITY AND MOLECULAR CHARACTERIZATION

    Direttore della scuola: Ch.mo Prof. Giuseppe ZANOTTI

    Coordinatore di Indirizzo: Ch.ma Prof.ssa Fiorella LO SCHIAVO

    Supervisore: Prof.ssa Patrizia POLVERINO DE LAURETO

    Dottoranda: Chiara FECCHIO

    A. A. 2013/2014

  • THESIS CONTENTS

    Riassunto............................................................................................................................ 1

    Summary............................................................................................................................ 5

    1. Introduction....................................................................................................................8

    1.1 Parkinson’s disease..........................................................................................8

    1.1.1 Pathogenesis 5

    Genetic factors in PD 6

    Park1: aS gene 17

    Post translational modification on aS 21

    Oxidative stress 19

    1.2 α-Synuclein.................................................................................................... 23

    1.2.1. Primary structure 24

    1.2.2. Secondary structure 26

    1.2.3. Mutations on aS gene 29

    1.2.4. Aggregation process 30

    1.2.5 Factors that influence aggregation of aS 33

    1.3 aS and lipids...................................................................................................36

    1.3.1 Structural features 36

    1.3.2 aS influence on lipid bilayers. 38

    1.3.3 Lipids influence on aS properties. 39

    1.3.4 Role of fatty acids 40

    2. Matherial and methods................................................................................................. 43

    2.1 Materials 43

    2.2 Methods 43

    2.2.1 Expression and purification of recombinant aS and aS mutants 43

    2.2.2 Aggregation studies 43

    2.2.3 Circular Dichroism 44

    2.2.4 Gel filtration 44

    2.2.5 RP-HPLC 44

    2.2.6 SDS-PAGE 45

  • 2.2.7 Native-PAGE 45

    2.2.8 Liposome preparation 46

    2.2.9 Thioflavin T binding assay (ThT) 46

    2.2.10 Dynamic Light Scattering (DLS) 46

    2.2.11 Transmission Electron Microscopy 46

    2.2.12 Mass spectrometry 46

    2.2.13 Proteolysis 47

    2.2.14 Ion exchange chromatography 47

    2.2.15 DNPH assay 47

    2.2.16 Atomic Force Microscopy 48

    2.2.17 Release assayes 48

    2.2.18 Cell permeabilization assay 48

    2.2.19 Planar Lipid membrane 49

    3. Results aS/DHA OLIGOMERS CHARACTERIZATION.......................................................51

    3.1 Aggregation process of aS in the presence of DHA and isolation

    of oligomers 51

    3.2 Chemico-physical characterization of aS/DHA oligomers 55

    4. Results INTERACTION WITH MEMBRANES....................................................................59

    4.1 Morphological analyses of liposomes 59

    3.2 aS/DHA oligomers interact with membranes 60

    3.3 Aggregation in the presence of lipids and membranes 62

    3.4 aS/DHA oligomers induce permeabilization of synthetic membranes 65

    4.5 aS/DHA oligomers permeabilize cellular membranes 67

    4.6 Mechanism of permeabilization of aS/DHA oligomers of membranes 68

    4.7 Pore formation 71

    5. 5. CHEMICAL MODIFICATIONS ON aS/DHA OLIGOMERS..............................................79

    5.1 DHA induces oxidative modifications on aS 79

    5.2 Detection of DHA covalent adduct on aS 83

    5.3 Role of His50 in oligomerization 86

    6. aS FAMILIAR VARIANTS AND DHA.................................................................................95

  • 6.1 Conformational analysis. 96

    6.2 Proteolytic mapping of the complex protein/DHA. 98

    6.3 Aggregation of as variants in the presence of DHA 102

    7. Discussion.....................................................................................................................111

    7.1 aS/DHA oligomers characterization 114

    7.2 aS/DHA oligomers activity on membranes 116

    7.3 Chemical modification of aS in the presence of DHA 118

    7.4 aS variants in the presence of DHA 120

    8. References....................................................................................................................123

    Published paper (Fecchio et al., 2013)

  • List of figures

    Figure 1.1 Loss of pigmented neurons in substantia nigra………………………………………........7

    Figure 1.2 Fibril formation leads to neuron cell death........................................................8

    Figure 1.3 Dopamine synthesis and pathway......................................................................9

    Figure 1.4 Pathways in PD pathogenesis............................................................................11

    Figure 1.5 Sequence of aS...................................................................................................14

    Figure 1.6 Methionine and tyrosine oxidation scheme......................................................18

    Figure 1.7 Schematic representation of aS structure......................................................... 24

    Figure 1.8 Schematic representation of aS aggregation process........................................30

    Figure 1.9 aS binding model to lipid vesicles......................................................………........37

    Figure 1.10 Metabolism of polyunsaturated fatty acids…..................................................41

    Figure 3.1 Gel filtration and RP-HPLC of aS/DHA mixture.………………………........................52

    Figure 3.2 IEX chromatography of aS (A) and aS/DHA 1:50 ………………………………….……….........54

    Figure 3.3 Far-UV CD spectra of monomeric and oligomeric aS..…………………………….........55

    Figure 3.4 Structural characterization of aS/DHA oligomers …………….........…………….........57

    Figure 4.1 DLS and TEM of SUV of different composition...................................................60

    Figure 4.2 Far-UV CD of aS/DHA oligomers in the presence of vesicles...…….………….........61

    Figure 4.3 Membrane damage caused by aggregation of aS on membrane......................62

    Figure 4.4 Aggregation studies monitored by ThT assay …………………….………………………….63

    Figure 4.5 Aggregation studies monitored by far-UV CD..……………..……….………..……….......64

    Figure 4.6 Calcein-leakage test on liposomes ……….....................................……….……………66

    Figure 4.7 PI influx in dopaminergic cells............................................................................67

    Figure 4.8 DLS measurement of oligomers effect on liposomes.........……………………..........69

    Figure 4.9 TEM of SUV in the absence and in the presence of aS/DHA oligomers.............70

    Figure 4.10 Schematic representation of PLM........................................................................71

    Figure 4.11 PLM experiments..................................................……………………….…..…..........71

    Figure 5.1 Schematic representation of reaction of DNPH on carbonyls.……………………………….79

    Figure 5.2 UV-Vis spectra of DNPH labeled aS....…………………………………………………………...81

  • Figure 5.3 RP-HPLC analyses of DNPH labeled aS.........................………..……………….……..…81

    Figure 5.4 RP-HPLC analyses of proteolysis of DNPH labeled aS..............……………………….82

    Figure 5.5 RP-HPLC analysis proteolysis of aS/DHA oligomers...........................................84

    Figure 5.6 MS-MS of peptide 46-58…………………..........................................................……..85

    Figure 5.7 Sequences of aS peptides with a Δ mass of 326 Da.….............…………..………..….85

    Figure 5.8 Purification of H50Q.................................................…………………………………......86

    Figure 5.9 CD spectra of aS and H50Q in the absence and in the presence of DHA...........87

    Figure 5.10 RP-HPLC of aS and H50Q proteolysis in the presence of DHA......…….…………..87

    Figure 5.11 Gel filt