Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Justin Legleiter

Committee Co-Chair

Fabien Goulay

Committee Member

Terry Gullion

Committee Member

Blake Mertz

Committee Member

Visvanathan Ramamurthy

Abstract

Huntington disease is a genetic, neurodegenerative disease caused by an expanded polyglutamine (polyQ) in the first exon of the huntingtin (htt) protein, facilitating its aggregation, leading to the formation of a diverse population of potentially toxic aggregate species, such as oligomers, fibrils, and annular aggregates. Htt interacts with a variety of membranous structures within the cell, and the first seventeen amino acids (Nt17) of htt directly flanking the polyQ domain is an amphipathic alpha-helix (AH) lipid-binding domain. AHs are also known to detect membrane curvature. Nt17 also promotes diverse aggregate species of htt and undergoes a number of posttranslational modifications that can modulate htt's toxicity, subcellular localization, and trafficking of vesicles. To get in-depth mechanistic insights of huntingtin aggregation, both chemical and physical modulators of the aggregation process were explored. Specifically, the importance of htt acetylation and the role of membrane curvature on htt aggregation were investigated using atomic force microscopy (AFM), which has become a robust technique to obtain physical insights into the formation of toxic protein aggregates associated with amyloid diseases on lipid membranes. Acetylation of htt exon 1, and synthetic truncated htt exon1 mimicking peptide (Nt 17Q35P10KK) was achieved using a selective covalent label sulfo-N-hydroxysuccinimide (NHSA) in molar ratios of 1x, 2x, and 3x NHSA per peptide. Htt acetylation was found to decrease fibril formation in solution and promoted the formation of larger globular aggregates. Acetylation strongly altered htt's ability to bind lipid membranes. However, one of the several limitations associated with using these current flat, supported bilayers as model surfaces is the absence of membrane curvature, which can heavily influence the interaction of proteins at lipid interfaces. Using an AFM force reconstruction technique, silicon substrate, and silica nanobeads, model lipid bilayer system was developed and validated in which the underlying solid support is comprised of flat and curved regions to induce regions of curvature in the bilayer. This model bilayer system was exposed to Nt17Q35P10KK peptide, and this peptide preferentially bound and accumulated to curved membranes, consistent with the ability of AHs to sense membrane curvature.

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