Graduation Year

2011

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Physics

Major Professor

Martin Muschol

Abstract

Formation of large fibers and plaques by amyloid proteins is recognized as the molecular hallmark of an increasing number of human disorders, including Parkinson's disease, Alzheimer's disease and even type II diabetes. The broader objective of my research is to unravel the basic mechanisms that initiate and regulate fibril formation by amyloidogenic proteins. This objective is significant because even basic aspects of how fibril formation proceeds from a soluble, monomeric protein to an insoluble amyloid fibril remain much debated. Furthermore, there is increasingly strong evidence suggesting that intermediates of the aggregation process, with properties distinct from those of mature fibrils, are the aggregate species most toxic to human tissues. Combining non-intrusive optical techniques (dynamic light scattering) to characterize the nucleation and growth kinetics of aggregation intermediates with high-resolution imaging (atomic force microscopy) to characterize aggregate morphologies and their physical dimensions, we have investigated the self-assembly process of two distinctly different amyloidogenic proteins - hen egg white lysozyme and human recombinant tau.

Initially, we used hen egg white lysozyme to characterize the kinetics and morphology of various intermediates emerging along a fixed assembly pathway. We further investigated whether and how the fibril assembly process for lysozyme changed as function of salt concentration. These experiments revealed that lysozyme displays three distinct types of aggregation behavior at different ionic strengths: monomeric fibril assembly, oligomeric fibril assembly, and amorphous precipitation. We followed these observations by exploring whether and how net intermolecular interactions among lysozyme monomers and the intramolecular conformation of lysozyme affect these transitions in the assembly process. We found that the prevailing intermolecular interactions played dominant roles in regulating fibril assembly pathways, suggesting that protein interactions hold critical clues on how to control amyloid fibril assembly.

Using the same experimental approaches, we investigated the role of heat shock proteins as regulators of tau fibril assembly. The native function of human tau is to stabilize the microtubules in the axonal processes of neurons. The accumulation of aberrant tau into neurofibrillar tangles is diagnostic of many neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Pick's disease. A significant body of research indicates that phosphorylation, oxidation, and ubiquitination of human tau can impair its affinity for microtubules and trigger self-assembly of hyperphosphorylated tau into these neurofibrillar tangles. Cells possess a large network of regulatory proteins called chaperones which are responsible for the proper folding and "disposal" of proteins, such as misfolded tau. We therefore investigated the role of different chaperones on in vitro fibril assembly of tau. We show that the small chaperone Hsp27, regardless of phosphorylation, is capable of inhibiting tau fibril growth in vitro. In contrast, dynamic phosphorylation and dephosphorylation of Hsp27 in vivo are necessary mechanisms for tau clearance and inhibition. By defining these differences in tau aggregation under in vitro vs. in vivo conditions, we hope to gain better understanding on how the chaperone network interacts with tau and how to target various chaperones for therapeutic interventions.

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