The structure and assembly of apolipoprotein C-II amyloid fibrils

© 2011 Dr. Chai Lean TeohThe self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein (apo) C-II forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. This project examines the formation of apoC-II amyloid fibrils, with a focus on its structure and assembly. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β structure, composed of two β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy and atomic force microscopy revealed that the fibrils are flat ribbons composed of one molecule per 4.7 Å rise of the cross-β structure. The ability of single cysteine substitution mutants to form cross-links is consistent with a parallel, in-register structural model for apoC-II fibrils, while fluorescence resonance energy transfer analysis of apoC-II fibrils provided distance constraints within the fibrils. On the basis of these studies, a simple structural model composed of stacked apoC-II subunits in a ‘letter G-like’ β-strand-loop-β-strand conformation was proposed. The time course of the molecular dynamics simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel-in-register models. The development of this model for apoC-II amyloid fibrils provided a basis for investigations into the factors that control the properties and stabilities of amyloid fibrils. The effects of amino acid substitutions, seeding, shear-flow and lipids on fibril assembly and morphology were examined. ApoC-II forms amyloid fibrils in a lipid-dependent manner via an initial nucleation step, where lipid induced rapid formation of a tetrameric species, which is followed by a slow isomerisation that precedes monomer addition and fibril growth. The ability of apoC-II pre-formed dimers to self-assemble into amyloid fibrils and act as seeds for apoC-II amyloid fibril formation, led to the proposal of an alternate fibril assembly pathway, which incorporates isomerization of monomers into a configuration that favours rapid self-assembly into mature fibrils. Studies on the effects of shear-flow on apoC-II amyloid fibril formation showed that shear induced an irreversible structural change in apoC-II monomers, which subsequently form an on-pathway pre-fibrillar oligomeric species. Lastly, the final chapter demonstrates the application of total internal reflection fluorescence microscopy to visualize the polymorphism in apoC-II amyloid fibrils, where low concentrations of micellar phospholipids and lipid bilayers induced a novel, straight rod-like morphology for apoC-II fibrils, which is distinct from the twisted-ribbon morphology observed for apoC-II fibrils formed in the absence of lipids. Overall, these studies shed light on the molecular organization of apoC-II in the amyloid form and add to an understanding of the molecular basis of amyloid formation and deposition by apolipoproteins. They also raise a number of potential areas for further investigation which will lead to an improved understanding of the interactions that stabilize amyloid structures, the commonalities and differences among amyloid structures, the mechanisms by which amyloid fibrils form, and potential practical uses of amyloid structures