Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways

Abstract

We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-alpha(2) octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and beta-sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

Does the Cytotoxic Effect of Transient Amyloid Oligomers from Common Equine Lysozyme in Vitro Imply Innate Amyloid Toxicity?

“…2 shows that the rings exhibit a clearly segmented structure, and the molecular dimensions of the segments are similar to the parameters of individual oligomers present in solution, which suggests that the rings can be formed from round-shaped oligomers. A similar morphology was observed in the amyloid rings of the de novo designed protein albebetin, which also demonstrate a well defined segmentation and are assembled from oligomers (31).…”

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

“…The compactness of this molecule is achieved due to short loops connecting the secondary structure elements. In our previous studies we have shown that albebetin readily assembles into a variety of amyloid structures during its incubation under physiological conditions [21]. Here we examine the cytotoxic properties of main amyloid species of albebetin relating their structural properties with exerted cytotoxicity.…”

Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status

“…8). The cytotoxicity of the soluble, oligomeric forms is dependent on the type of oligomeric species [153][154][155][156][157][158][159], and existence of both on-and off-pathway oligomers has been proposed [20,60,150,153,160]. Hydrophobic probes such as 8-anilino-1-naphthalenesulfonic acid and the molecular rotor 9-dicyanovinyl-julolidine have been shown to respond to earlier species such as TTR oligomeric species [60].…”