The functional studies of amyloid fibrils and their toxicity

Abstract

Amyloid fibrils are a form of highly ordered, β-sheet protein structure found in many sites in the body. Fibril formation occurs when intermediates along the protein-folding pathway irreversibly enter the off-folding pathway to form highly ordered amyloid fibrils. Amyloid fibril formation is of considerable research interest because of its intimate association with a wide range of debilitating diseases, including Alzheimer’s, Huntington’s and Parkinson’s diseases and type II diabetes. Recently, it has been found that amyloid fibrils enhance human immunodeficiency virus (HIV-1) infection. Semen contains a fibril forming component that significantly increases the ability of HIV-1 to infect cells. This component is associated with peptide fragments of prostatic acid phosphatase and has been termed semen-derived enhancer of viral infection (SEVI). SEVI acts at the virus entry stage and only boots infectivity when the peptide has folded into highly structured arrays of amyloid fibrils. The work presented in this thesis describes the broader roles of SEVI in HIV-1 infection including its toxicity to neuronal and epithelial cells as well as the toxicity of αs2-casein [s2 subscript] (milk fibril forming protein). Firstly, the amyloidogenic regions of SEVI are identified by the use of computer algorithms. Accordingly, these regions were synthesised to examine their individual fibril-forming propensity. Fragments from the central regions formed fibrils of similar morphology to SEVI at physiological pH and temperature. Fibril formation was assessed via thioflavin T assay, circular dichroism spectrometry and transmission electron microscopy (TEM). In this study the toxicity of SEVI and its amyloidogenic fragments to neuronal and epithelial cells was investigated. SEVI and its fragments were toxic to neuronal cells but not to confluent epithelial cells. Secondly, the coreceptors used by SEVI and its amyloidogenic fragments are identified. HIV enters the cell by the interaction of glycoprotein (gp) 120 envelope with the cellular differentiation (CD) 4 protein and secondary coreceptors. Affinofile assays showed that SEVI and its fragments use CCR5 and CXCR4 secondary coreceptors to enhance HIV-1 entry to the host cells. Additionally the ability of clusterin to inhibit fibril formation by SEVI was investigated. Clusterin inhibited fibril formation by SEVI in a concentration dependent manner thereby inhibiting the cytotoxicity associated with the fibrils. Lastly, work detailing the toxicity of fibrils formed by milk protein αs2-casein [s2 subscript] is presented. αs2-casein [s2 subscript] forms fibrils spontaneously under physiological conditions. These fibrils have been found in corpora amylacea, an amyloid condition that infrequently develops within the mammary tissue of cows. The use of cell toxicity assays show that fibrils formed by αs2-casein [s2 subscript] were toxic to pheochromocytoma (PC) 12 cells. Furthermore, the use of Thioflavin T assay and TEM showed that a polyphenol epigallocatechin-3-gallate (EGCG), a component found in green tea extracts, inhibits fibril formation by αs2-casein [s2 subscript]. Previous research has found that, EGCG can reduce fibril formation and cellular toxicity of various fibril-forming proteins. EGCG has also been shown to inhibit SEVI enhancement of HIV infection in a manner dependent on the ability of EGCG to disrupt SEVI fibril formation, providing proof of principle for the potential of anti-fibril agents as inhibitors of HIV infection.