Amyloid Conformational Diversity Accessed By Truncations of a Native Protein Fold 公开

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

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
关键词	self-assembly nucleation amyloid
Committee Chair / Thesis Advisor	Lynn, David, Emory University
Committee Members	Lutz, Stefan, Emory University Dyer, Brian, Emory University

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