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Plasma apolipoproteins form amphipathic a helices in lipid environments but in the lipid-free state show a high propensity to form β structure and self-associate into amyloid fibrils. The widespread occurrence of apolipoproteins in amyloid plaques suggests disease-related roles, specifically in atherosclerosis. To reconcile the dual abilities of apolipoproteins to form either a helices or cross-β sheet structures, we examined fibrils formed by human apolipoprotein C-II (apoC-II). A structural model for apoC-II fibrils shows a cross-β core with parallel ft strands, including a buried K30-D69 charge pair. We investigated the effect of abolishing this charge pair in mutant D69K apoC-II. Fluorescence studies indicated more rapid fibril formation and less solvent accessibility of tryptophan (W26) in D69K apoC-II fibrils than in wild-type (WT) fibrils. X-ray diffraction data of aligned D69K apoC-II fibrils yielded a typical cross-β structure with increased ft sheet spacing compared to that of WT fibrils. Hydrogen/deuterium (H /D) exchange patterns were similar for D69K apoC-II fibrils compared to WT fibrils, albeit with an overall reduction in the level of slow H /D exchange, particularly around residues 29--32. Molecular dynamics simulations indicated reducedβ strand content for a model D69K apoC-II tetramer compared to the WT tetramer and confirmed an expansion of the cross-β spacing that contributed to the formation of a stable charge pair between K69 and E27. The results highlight the importance of charge-pair interactions within the apoC-II fibril core, which together with numerous salt bridges in the flexible connecting loop play a major role in tire ability of lipid-free apoC-II to form stable cross-β fibrils. [ABSTRACT FROM AUTHOR]

Protein misfolding and aggregation, leading to Amyloid fibrils amyloid fibril formation, are characteristic of many devastating and debilitating amyloid diseases. Accordingly, there is significant interest in the mechanisms underlying amyloid fibril formation and identification of possible intervention tools. Small molecule drug compounds approved for human use or for use in phase I-III clinical trials were investigated for their effects on amyloid formation by human apolipoprotein (apo) C-II. Several of these compounds modulated the rate of amyloid formation by apoC-II. Epigallocatechin gallate (EGCG), a green tea catechin, was an effective inhibitor of apoC-II fibril formation, and the antipsychotic drug, fluphenazine-HCl, was a potent activator. Both EGCG and fluphenazine-HCl exerted concentration-dependent effects on the rate of fibril formation, bound to apoC-II fibrils with high affinity, and competitively reduced thioflavin T binding. EGCG significantly altered the size distribution of fibrils, most likely by promoting the lateral association of fibrils and subsequent formation of large aggregates. Fluphenazine-HCl did not significantly alter the size distribution of fibrils, but it may induce the formation of a small population of rod-like fibrils that differ from the characteristic ribbon-like fibrils normally observed for apoC-II. The findings of this study emphasize the effects of small molecule drugs on the kinetics of amyloid fibril formation and their roles in determining fibril structure and aggregate size. [ABSTRACT FROM AUTHOR]

The misfolding, aggregation, and accumulation of proteins as amyloid fibrils is a defining characteristic of several debilitating diseases. Human apolipoprotein C-II (apoC-II) amyloid fibrils are representative of the fibrils formed by a number of plasma apolipoproteins implicated in amyloid-related disease. Previous structural analyses identified a buried charge pair between residues K30 and D69 within apoC-II amyloid fibrils. We have investigated the effects of amino acid substitutions of these residues on apoC-II fibril formation. Two point mutations of apoC-II, D69K and K30D, as well as a reversed ion-pair mutant containing both mutations (KDDK) were generated. Fibril formation by the double mutant, apoC-II KDDK, and apoC-II D69K was enhanced compared to that of wild-type (WT) apoC-II, while apoC-II K30D lacked the ability to form fibrils under standard conditions. Structural analyses showed that WT apoC-II, apoC-II D69K, and apoC-II KDDK fibrils have similar secondary structures and morphologies. Size distribution analyses revealed that apoC-II D69K fibrils have a broader range of fibril sizes while apoC-II KDDK fibrils showed an increased frequency of closed fibrillar loops. ApoC-II D69K fibrils exhibited reduced thioflavin T binding capacity compared to that of fibrils formed by WT apoC-II and apoC-II KDDK. These results indicate that specific charge and charge-pair mutations within apoC-II significantly alter the ability to form fibrils and that position 69 within apoC-II plays a key role in the rate-limiting step of apoC-II fibril formation. [ABSTRACT FROM AUTHOR]