Structural Investigation of Helical Intermediates in the Misfolding Pathway of Amyloid Peptides Associated With Type II Diabetes and HIV.

Abstract

A variety of aging related diseases including Alzheimer’s, Type II diabetes, Huntington’s, Parkinson’s, and Creutzfeldt-Jakob are characterized by the formation of abnormal proteinaceous deposits. These deposits, known as amyloid, are abnormal accumulations of misfolded proteins with a characteristic cross β-sheet conformation. The formation of amyloid fibers can occur through many pathways, of which one of the most important pathway is catalyzed by binding to the cell membrane. The peptide-membrane interaction can disrupt the integrity of the cell membrane, causing disruption of calcium homeostasis and eventual cell death. In membrane-mediated aggregation, the protein is thought to initially bind with the membrane in an α-helical conformation before undergoing a conformational change during aggregation to a β-sheet form characterstic of the amyloid fiber. Therefore, it is important to determine atomic-level structures of these intermediates, as they can provide insights into the toxic mechanism exhibited by these amyloidogenic proteins and into the design of drugs that can suppress these intermediate helical species to stop further progression into toxic states. This dissertation reports high-resolution NMR structural studies of two different membrane bound amyloid proteins: (1) IAPP (an amyloidogenic peptide related to Type II diabetes), in order to understand the role of α-helical intermediate structures in causing membrane disruption, (2) PAP248-286 (the corresponding amyloid fiber (SEVI) enhances the infectivity of the HIV virus), in order to understand the bridging interactions it exhibits between the host and viral cell membranes. Our studies on the membrane bound α-helical intermediates of rat and human IAPP/IAPP1-19 reveal a pH dependent membrane orientation for IAPP1-19, which correlates well with its ability to disrupt synthetic membrane vesicles and β-cells, and that the position of the disulfide bridge with respect to the hydrophobic interface of the N-terminal helix could be one of the factors that modulate the membrane disruptive behaviour of these peptides. Our study on membrane bound PAP248-286, reveals an unusual amount of structural disorder that, in combination with high positive charge at the N-terminus could play an important role in the fusion of host and viral cell membranes by weakening the electrostatic interactions that repel similarly charged membranes.