Abstract
Neurodegenerative diseases have gained more attention in
the last decade due in part to advances in genomic information,
structural definition and imaging technology. The development of
devastating neurodegenerative conditions, such as Alzheimer's
disease (AD), is associated with the deposition of amyloid fibers
and neuronal apoptosis. These fibers are predominantly comprised of
the amyloid β peptide,
Aβ(1-42), and its
truncations, Aβ(1-40) and
Aβ(1-39). Recently,
Aβ amyloid formation in retinal
diseases such as glaucoma and macular degeneration have been shown
to eventually lead to neuronal cell death and subsequent
irreversible blindness, and recent data has correlated an increase
in development of glaucoma or macular degeneration of patients with
AD. Although many theories have been postulated regarding the true
cause of these retinal diseases, including increased intra-ocular
pressure and oxidative stress resulting in mitochondrial damage and
the release of cytochrome c, the relationship between AD and
glaucoma has only recently been investigated. Genomic studies of
patients with the most common form of glaucoma has shown that
mutations in three particular genes highly expressed in the ocular
tissues (optineurin, myocilin, and WDR36) result in
pre-glaucomatous conditions that degrade the optic nerve over time.
WDR36 has a membrane-bound
β-propeller and, similar to the well-known
signal transduction associated
β-propeller G-protein, each propeller blade
is made of a repeating WD-unit that folds into a
β-meander composed of four strands. My
hypothesis is that β-propeller
structures, such as the β subunit of
the G Protein and WDR36, provide a nucleation site for converting
the Aβ peptide into amyloid fibers
resulting in damage to retinal ganglion cells. Here, I show that
subunits and β-strand fragments of
the β-propeller fold modulate the
morphology Aβ(16-22), the nucleating
core of the amyloid β peptide,
allowing for the self-assembly of diverse conformations. These
observations may provide insight to both the tissue-specificity of
amyloid strains as well as the heterogeneous nature of the amyloid
deposits found in the disease state. Understanding sequence
contributions and having the ability to control the morphology and
dimensions of self-assembling peptide nanostructures has relevance
to disease etiology, development of therapeutics and the design of
future bio-inspired nanomaterials.
Table of Contents
Table of Contents
Acknowledgements
List of Figures
List of Tables
Abbreviations
Chapter 1: Protein Misfolding and Amyloid Tissue Specificity
Self-assembly is a Hallmark of Living Systems
............................................. 1
Glaucoma: An Ocular Amyloidosis
............................................................
3
Amyloid Assembly is Context Dependent
................................................... 8
Chapter 2: Models of a Glaucoma-Related β-Propeller Fold
Introduction
.............................................................................................
17
Results
.....................................................................................................
22
Building Homology Models of Proteins Implicated in Glaucoma
.......... 22
Homology within the β Propellers of G Proteins and WDR36 Model
.... 35
Mutations Destabilize the β-Propeller Fold
.......................................... 41
Discussion
................................................................................................
46
Materials and Methods
............................................................................
49
Chapter 3: β-Propeller Fragment Assembly and Modulation of
Self-Assembly in Peptide Chimeras
Introduction
.............................................................................................
52
Results
.....................................................................................................
57
Blade Fragments of G Protein Self-Assemble into
Amyloid................... 57
Aβ(16-22) Assembly is Attenuated in β-Hairpins
.................................. 67
Linker Sequence Affects the Morphology of Aβ(16-22) Chimeras
.......... 74
Discussion
................................................................................................
77
Materials and Methods
............................................................................
81
Chapter 4: β-Propeller Fragments Self-Assemble into Distinct
Morphologies
Introduction
.............................................................................................
85
Results
.....................................................................................................
87
β-propeller Fragments from the GPBS Self-Assemble
............................ 87
Characterization of the Spherical Peptide Particles
............................... 94
Discussion
...............................................................................................
104
Materials and Methods
...........................................................................
107
Chapter 5: β-Propeller Fragments Alter the Morphology of
Aβ(16-22)
Introduction
............................................................................................
111
Results
....................................................................................................
112
Aβ(16-22) Monomers Interact with Preassembled GPBS
β-Strands ...... 112
RLLLSA Monomers Do Not Seed Aβ(16-22) Tubes
............................ 120
RLLLSA Particles Transform Mature Aβ(16-22)
Fibrils....................... 121
Discussion
...............................................................................................
129
Materials and Methods
...........................................................................
134
Chapter 6: Peptide Particles Allow for Extended Lamination of
Aβ(16-22)
Introduction
............................................................................................
138
Results
....................................................................................................
142
RLLLSA Particles Remove Rhodamine Dye from Solution
................. 142
Biotin-labeled RLLLSA Concentrates Strepavidin-GNP
...................... 144
RLLLSA Does Not Change the Morphology of Aβ(16-22) E22L
......... 147
Post-Freeze TEM and Fluorescence Microscopy Suggest a Nucleation
Event........ 151
IE-FTIR Shows No RLLLSA Incorporation into the Mixed
Nanotubes............. 161
Discussion
...............................................................................................
167
Materials and Methods
...........................................................................
172
Chapter 7
Conclusions
............................................................................................
179
Peptide Sequence and Conformation
................................................ 179
β-Propeller Folds: Platforms for Peptide-Peptide Interactions
............ 180
Learning the Rules of Self-Assembly
.................................................. 181
The Shallow Energy Landscape for Amyloid Assembly
.................... 182
Appendix I
......................................................................................................
187
References
.......................................................................................................
191
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