UC Irvine

Chapter 1 provides an overview of the field of amyloid structural biology and provides context for the work described in this dissertation. In the more than a century since its identification, Alzheimer’s disease has become the archetype of amyloid diseases. The first glimpses of the chemical basis of Alzheimer’s disease began with the identification of “amyloid” plaques in the brain in 1892 and extended to the identification of proteinaceous fibrils with “cross-β” structure in 1968. Further efforts led to the discovery of the β-amyloid peptide Aβ as a 40- or 42-amino acid peptide that is responsible for the plaques and fibrils. At this point, a three-decade long marathon began to elucidate the structure of the fibrils and identify the molecular basis of Alzheimer’s disease. Along the way, an alternative model began to emerge in which small aggregates of Aβ, called “oligomers”, rather than fibrils, are the culprits that lead to neurodegeneration in Alzheimer’s disease. This dissertation describes my efforts to understand the structural, biophysical, and biological properties of the oligomers in Alzheimer’s disease.

β-Sheets are the building blocks of amyloid fibrils and oligomers. Amyloid fibrils generally consist of extended networks of parallel β-sheets. Amyloid oligomers appear to be more compact enclosed structures, some of which are thought to be composed of antiparallel β-sheets comprising β-hairpins. β-Hairpins are special because their twisted shape, hydrophobic surfaces, and exposed hydrogen-bonding edges impart a unique propensity to form compact assemblies. Our laboratory has developed macrocyclic β-sheets that are designed to mimic β-hairpins formed by amyloidogenic peptides and proteins. The β-hairpin mimics contain two β-strand peptide fragments linked together at their N- and C-termini by two δ-linked ornithine turn mimics to create a macrocycle. An N-methyl group is installed on one of the β-strands to prevent uncontrolled aggregation. These design features facilitate crystallization of the β-hairpin mimics and determination of the X-ray crystallographic structures of the oligomers that they form.

During the past few years, our laboratory has elucidated the X-ray crystallographic structures of oligomers formed by β-hairpin mimics derived from Aβ, α-synuclein, and β2-microglobulin. Out of these three amyloidogenic peptides and proteins, the Aβ β-hairpin mimics have provided the most insight into amyloid oligomers. Our studies have revealed a previously undiscovered mode of self-assembly, whereby three Aβ β-hairpin mimics assemble to form a triangular trimer. The triangular trimers are remarkable, because they contain two largely hydrophobic surfaces that pack together with other triangular trimers to form higher-order oligomers, such as hexamers and dodecamers. Some of the dodecamers pack in the crystal lattice to form annular porelike assemblies. Some of the β-hairpin mimics and triangular trimers assemble in solution to form oligomers that recapitulate the crystallographically observed oligomers. These oligomers exhibit toxicity toward neuronally derived cells, recapitulating the toxicity of the oligomers formed by full-length amyloidogenic peptides and proteins. These findings are significant, because they address a gap in understanding the molecular basis of amyloid diseases. We anticipate that these studies will pave the way for developing diagnostics and therapeutics to combat Alzheimer’s disease, Parkinson’s disease, and other amyloid diseases

Chapter 2 presents the X-ray crystallographic structures of oligomers formed by a 20-residue peptide segment derived from Aβ. The development of a peptide, in which Aβ17–36 is stabilized as a β-hairpin is described and the X-ray crystallographic structures of oligomers it forms are reported. Two covalent constraints act in tandem to stabilize the Aβ17–36 peptide in a hairpin conformation: a δ-linked ornithine turn connecting positions 17 and 36 to create a macrocycle, and an intramolecular disulfide linkage between positions 24 and 29. An N-methyl group at position 33 blocks uncontrolled aggregation. The peptide readily crystallizes as a folded β-hairpin, which assembles hierarchically in the crystal lattice. Three β-hairpin monomers assemble to form a triangular trimer, four trimers assemble in a tetrahedral arrangement to form a dodecamer, and five dodecamers pack together to form an annular pore. This hierarchical assembly provides a model, in which full-length Aβ transitions from an unfolded monomer to a folded β-hairpin, which assembles to form oligomers that further pack to form an annular pore. This model may provide a better understanding of the molecular basis of Alzheimer’s disease at atomic resolution.

Chapter 3 describes the design, synthesis, X-ray crystallographic structures, and biophysical and biological properties of two stabilized trimers derived from Aβ17–36. These triangular trimers are stabilized through three disulfide crosslinks between the monomer subunits. The X-ray crystallographic structures reveal that the stabilized trimers assemble hierarchically to form hexamers, dodecamers, and annular porelike structures. Solution-phase biophysical studies reveal that the stabilized trimers assemble in solution to form oligomers that recapitulate some of the higher-order assemblies observed crystallographically. The stabilized trimers share many of the biological characteristics of oligomers of full-length Aβ, including toxicity toward a neuronally derived human cell line, activation of caspase-3 mediated apoptosis, and reactivity with the oligomer-specific antibody A11. These studies support the biological significance of the triangular trimer assembly of Aβ β-hairpins and may offer a deeper understanding of the molecular basis of Alzheimer’s disease.

Chapter 4 describes the design and study of a macrocyclic β-hairpin peptide derived from Aβ16–36. SDS-PAGE and size exclusion chromatography studies show that the Aβ16–36 β-hairpin peptide assembles in solution to form hexamers, trimers, and dimers. X-ray crystallography reveals that the peptide assembles to form a hexamer in the crystal state and that the hexamer is composed of dimers and trimers. LDH release assays show that the oligomers formed by the Aβ16–36 β-hairpin peptide are toxic toward neuronally derived SH-SY5Y cells. Replica exchange molecular dynamics (REMD) demonstrates that the hexamer can accommodate full-length Aβ. These findings expand our understanding of the structure, solution-phase behavior, and biological activity of Aβ oligomers, and may offer insights into the molecular basis of Alzheimer’s disease.