Fibrillogenesis and Cytotoxic Activity of the Amyloid-forming Apomyoglobin Mutant W7FW14F

Abstract

The apomyoglobin mutant W7FW14F forms amyloidlike fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing ␤-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Cytotoxicity of albebetin oligomers depends on cross‐β‐sheet formation

“…The second type includes oligomers of 25-40-mers size; their binding to amyloid specific dyes thioflavine T and Congo red and the development of far UV CD spectrum similar to the fibrillar CD signal indicate the formation of cross-b-sheet structure. Both types of oligomers exhibit similar roundshaped morphology in AFM images which is commonly associated with prefibrillar amyloid [1][2][3][4][5]. Here we have demonstrated that only the second enlarged type of oligomers produce cytotoxic effect on two kinds of neuronal cells.…”

“…However the definition of the toxic amyloid species and their particular structural properties exerting the specific cytotoxic effect remain controversial. In the last years a number of studies have supported the hypothesis that early aggregates or prefibrillar species are implicated in cytotoxicity while fibrillar amyloid deposits serve as an escape route for smaller aggregates [1][2][3][4][5]. Among them Kayed et al observed that intermediate-size water soluble oligomers of Ab and other five proteins form sheared structures characterized by similar conformational epitope, suggesting that they exert cytotoxicity via similar mechanisms [6].…”

The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

“…Despite years of research on the eukaryotic amyloids, curli are the only known amyloid pathway to have a dedicated nucleator protein in vivo. Mounting evidence suggests that amyloid precursors are cytotoxic, whereas the fibers are an innocuous final product of the misfolding event (38)(39)(40)(41)(42)(43)(44)(45)(46). Functional amyloidosis may have evolved specialized mechanisms to eliminate the build up of the cytotoxic intermediates.…”

Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates

“…Our study sought to provide information on this topic by exposing a panel of normal or pathological cell lines to toxic pre-fibrillar aggregates of HypF-N, a protein not associated with any amyloid disease, previously shown to display the same structural features and properties of the aggregates produced by disease-associated peptides and proteins (Relini et al, 2004). Previous reports have shown that pre-fibrillar aggregates of proteins not associated with disease are toxic to cells in a manner similar to early aggregates of disease-related proteins and peptides (Bucciantini et al, 2002;Kranenburg et al, 2003;Sirangelo et al, 2004) by impairing the same biochemical parameters . Under our experimental conditions, differing cell lines were variously affected by the same amount of toxic prefibrillar aggregates of a particular protein, whereas mature fibrils were substantially non-toxic; this finding suggests that the severity of cell impairment was related to the different ability of any cell line to counteract the biochemical modifications resulting from exposure to the aggregates.…”