TABLE OF CONTENTS
Acknowledgements
i
Abbreviations
ii
Abstract
iv
Conference proceedings
x
Foreword
xi
CHAPTER 1: INTRODUCTION
1.1 Diabetes
1
1.2 Structure of the IAPP gene
5
1.3 Biosynthesis of IAPP
7
1.4 Functions of IAPP
8
1.5 Amyloid formation
9
1.6 Fibril structure
13
1.7 Species-specific differences in the sequence of IAPP
15
1.8 Structural models for IAPP amyloid
17
1.9 What prevents IAPP from forming amyloid normally?
22
1.10 Why does IAPP amyloid form in Type 2 diabetes?
23
1.11 Defence against amyloidosis
26
1.12 Clusterin
28
CHAPTER 2: MATERIALS AND METHODS
2.1 MATERIALS
31
2.1.1 Buffers and solutions
33
2.1.2 Protein and antibodies
34
2.1.3 Bacterial culture media
34
2.1.4 Bacterial strains
34
2.2 METHODS
35
2.2.1 Agarose gel electrophoresis
35
2.2.2 Preparation of competent E. coli cells
35
2.2.3 Transformation of E. coli
35
2.2.4 Purification of pET 32/trx-proIAPP plasmid
36
2.2.6 SDS-PAGE
37
(a) Sample preparation
37
(b) Tris-glycine SDS-PAGE
37
(c) Tris-tricine SDS-PAGE
38
2.2.7 Cell lysis protocols for the purification under native conditions
(a) Lysis by sonication in the absence of detergents
38
(b) Cell lysis by incubation with 1% Triton X-100 in the presence
of PMSF
39
(c) Cell lysis by sonication in the presence of Triton X-100 and/or
sarcosyl with protease inhibitors
39
2.2.8 Purification of trx-proIAPP under denaturing conditions using Ni-NTA
chromatography
(a) Purification in 6 M urea
39
(b) Purification 6 M GuHCl using Ni-NTA chromatography and
refolding by dilution
40
(c) Purification in 6 M GuHCl by Ni-NTA chromatography and
refolding on the Ni-NTA column
41
2.2.9 RP-HPLC protocols
(a) Purification of trx-proIAPP
41
(b) Purification of proIAPP from trx-tag
42
2.2.10 Enterokinase cleavage conditions
(a) Optimization trials
42
(b) Optimized enterokinase cleavage conditions
43
2.2.11 Electrospray mass spectrometry
43
2.2.12 Purification of proIAPP from trx-tag using Ni-NTA chromatography
43
2.2.13 Removal of buffer salts and reconcentration of proIAPP using a
Strata-X column
43
2.2.14 Reconstitution of proIAPP lyophilisate
44
2.2.15 Circular dichroism spectropolarimetry
44
2.2.16 Fluorescence assays
(a) ThT assays
45
(b) Bis-ANS assays
46
2.2.17 Congo Red Spectral Assay
47
2.2.18 Clusterin purification
47
2.2.19 Transmission electron microscopy aggregation assay
48
2.2.20 TEM and immunogold labelling of clusterin
48
2.2.21 Quantitation of clusterin binding to proIAPP by a direct ELISA
49
2.2.22 Quantitation of clusterin binding to proIAPP by competition ELISA
50
2.2.23 Bicinchoninic acid assay
51
2.2.24 Zone electrophoresis and amido black staining and immunoblotting
for clusterin
52
2.2.25 Dot blotting
53
2.2.26 General culture conditions for RIN-m5f cells
54
2.2.27 Cytotoxicity of proIAPP measured by MTS
54
2.2.28 Controls for cytotoxicity assays
55
2.2.29 The effect of adding clusterin to proIAPP at different stages of
amyloid formation on cytotoxicity
55
2.2.30 The cytotoxicity of proIAPP species formed in the presence and absence
of clusterin
56
2.2.31 ProIAPP binding to heparin-agarose beads
56
2.2.32 FITC-labelling of heparin
57
2.2.33 Measurement of the FITC-heparin associated with fibrillar proIAPP
58
CHAPTER 3: OPTIMIZATION OF ProIAPP PURIFICATION
3.1 INTRODUCTION
59
3.2 RESULTS
60
3.2.1 Previous methods for proIAPP purification
60
3.2.2 Overexpression of trx-proIAPP in E. coli BL21 cells
60
3.2.3 Attempts to solubilise trx-proIAPP
61
3.2.4 Purification of trx-proIAPP in 6 M urea using Ni-NTA
chromatography
3.2.5 Further purification of trx-proIAPP by RP-HPLC
65
66
3.2.6 Purification of trx-proIAPP in 6 M GuHCl using Ni-NTA
chromatography and refolding by dilution
68
3.2.7 Purification of trx-proIAPP in 6 M GuHCl by Ni-NTA
chromatography and refolding on the Ni-NTA column
70
3.2.8 Optimization of enterokinase cleavage conditions
71
3.2.9 Purification of proIAPP from trx-tag by RP-HPLC
74
3.2.10 Purification of proIAPP from trx-tag using Ni-NTA chromatography
76
3.2.11 Removal of buffer salts and re-concentration of proIAPP using a
Strata-X column
3.5 DISCUSSION
77
79
CHAPTER 4: ProIAPP AMYLOID FORMATION
4.1 INTRODUCTION
83
4.2 RESULTS
4.2.1 Secondary structure of proIAPP
85
4.2.2 Ageing of proIAPP followed by far-UV-CD
89
4.2.3 Kinetics of proIAPP amyloid formation
91
4.2.4 The effect of macromolecular crowding
94
4.2.5 The effect of ionic strength on proIAPP amyloid formation
96
4.2.6 SDS-PAGE analysis of proIAPP amyloid formation
97
4.2.7 Binding of Congo Red to proIAPP fibrils
100
4.2.8 Transmission electron microscopy of proIAPP fibrils
101
4.2.9 Binding of bis-ANS to proIAPP during amyloid formation
104
4.3 DISCUSSION
108
4.3.1 Structure of native proIAPP
108
4.3.2 Formation of amyloid from proIAPP
108
4.3.3 Structural changes of proIAPP during amyloid formation
110
CHAPTER 5: THE EFFECT OF CLUSTERIN ON ProIAPP AMYLOID
FORMATION AND CYTOTOXICITY
5.1 CYTOTOXICITY ASSOCIATED WITH AMYLOID FORMATION
112
5.1.1 The effect of IAPP amyloid formation on -cell destruction
112
5.1.2 Amyloidogenic intermediates are the toxic species
113
5.1.3 The ion channel hypothesis
113
5.1.4 The role of Ca2+ in cytotoxicity
115
5.1.5 Modulators of pore formation
116
5.1.6 The oxidative stress hypothesis
117
5.1.7 Association of ROS with Ca2+ influx
119
5.1.8 Cell death may occur by apoptosis or necrosis
120
5.2 THE ROLE OF CHAPERONES IN AMYLOID DISEASES
120
5.2.1 Clusterin and Alzheimer’s disease
120
5.2.2 Clusterin and other amyloidoses
123
5.2.3 Clusterin and Type 2 diabetes
124
5.2.4 Aims of this study
124
5.3 RESULTS
5.3.1 The effect of clusterin on amyloid formation measured by ThT assays
126
5.3.2 Analysis of proIAPP and clusterin mixtures by transmission electron
microscopy
128
5.3.3 The binding of clusterin to freshly redissolved proIAPP measured by
direct ELISA
131
5.3.4 The binding of clusterin to freshly redissolved proIAPP by competition
ELISA
133
5.3.5 The binding of clusterin to different denatured proteins in the
presence of proIAPP at pH 7.4
137
5.3.6 The binding of clusterin to different denatured proteins in the
presence of proIAPP at pH 5.5
141
5.3.7 Time course of binding of clusterin to denatured lysozyme in the
presence of proIAPP
144
5.3.8 The effect of clusterin on proIAPP amyloid formation monitored
by CD spectropolarimetry
5.3.9 The formation of complexes between clusterin and proIAPP
147
148
5.3.10 Dot-blotting for clusterin in soluble and insoluble fractions of proIAPP
and clusterin mixtures
5.3.11 Binding of clusterin to mature proIAPP fibrils
153
158
5.3.12 Clusterin bound to proIAPP fibrils detected by immunoelectron
microscopy
160
5.4 THE EFFECTS OF CLUSTERIN ON ProIAPP CYTOTOXICITY
5.4.1 The experimental design of cytotoxicity experiments
163
5.4.2 Cytotoxicity of different proIAPP species to a RIN-m5f cells
165
5.4.3 The concentration-dependence of proIAPP cytotoxicity
168
5.4.4 The effect of clusterin on proIAPP cytotoxicity
168
5.4.5 The cytotoxicity of proIAPP species formed in the presence of clusterin
170
5.5 DISCUSSION
5.5.1 The effects of clusterin on proIAPP amyloid formation
174
(a) The inhibition of amyloid formation
174
(b) ELISA measurements of the binding of clusterin to proIAPP
174
(c) Zone electrophoresis analysis
175
(d) CD analysis
176
(e) The interaction between clusterin and soluble proIAPP species
176
(f) The binding of clusterin to insoluble proIAPP species
178
5.5.2 The effects of clusterin on proIAPP-mediated cytotoxicity
180
5.5.3 The role of clusterin in Type 2 diabetes
182
CHAPTER 6: THE EFFECTS OF GLYCOSAMINOGLYCANS ON
ProIAPP AMYLOID FORMATION
6.1 INTRODUCTION
6.1.1 The localization of proteoglycans to amyloid deposits
183
6.1.2 The accumulation of proteoglycans in amyloid diseases
184
6.1.3 The role of proteoglycans in amyloid formation
185
6.1.4 GAGs may increase the stability of amyloid
186
6.1.5 Conformational changes induced by GAGs
187
6.1.6 Determinants of GAG binding to amyloidogenic peptides
188
6.1.7 The effect of GAGs on the rate of amyloid formation in vitro
191
6.1.8 GAGs and proIAPP
192
6.1.9 Aims of this study
194
6.2 RESULTS
6.2.1 The effect of GAGs on proIAPP amyloid formation
196
6.2.2 The effect of GAGs on the secondary structure of proIAPP
198
6.2.3 The concentration-dependence of heparin-induced conformational
changes in proIAPP
201
6.2.4 The concentration-dependence of heparin-induced amyloid formation
at pH 7.4 and 5.5
6.2.5 The mechanism of the heparin enhancement of ThT fluorescence
203
206
6.2.6 Metal cation dependence of heparin-induced amyloid formation
208
6.2.7 The effect of heparins of different molecular weights on
formation of amyloid from proIAPP
210
6.2.8 The effect of heparin disaccharides on heparin-induced proIAPP
amyloid formation
213
6.2.9 The effect of heparin on the exposed hydrophobic surface of
proIAPP measured by bis-ANS fluorescence
216
6.2.10 Changes in the fluorescence of bis-ANS bound to proIAPP during
heparin-induced proIAPP amyloid formation
218
6.2.11 The binding of soluble proIAPP to heparin-agarose
221
(a) The binding of proIAPP to heparin-agarose in
10 mM sodium phosphate, pH 7.4
(b) The binding of proIAPP to heparin-agarose in PBS, pH 7.4
222
223
6.2.12 Conformational changes of proIAPP induced by heparin in
10 mM sodium phosphate, 150 mM NaF, pH 7.4.
6.2.13 Incorporation of FITC-heparin into proIAPP fibrils
224
227
6.3 DISCUSSION
6.3.1 The effect of different GAGs on proIAPP amyloid formation
230
6.3.2 The minimal subunit of heparin necessary for proIAPP binding
231
6.3.3 The effect of buffers of differing ionic strength on heparin-induced
conformational changes in proIAPP
6.3.4 The effect of heparin on the final ThT fluorescence of proIAPP amyloid
CHAPTER 7: REFERENCES
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