Anand, B. G. | Search bioRxiv

bioRxiv2022

An efficient method to generate fluorescent amyloid fibrils

Studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis including their cellular internalization and crosstalk potential. Despite several advantages, unavoidable difficulties including expensive and tedious synthesis-protocols exist in fluorophore-based tools. Importantly, covalently-tagged fluorophores could introduce structural constraints which may influence the conformation of the monomeric and aggregated forms of protein. To resolve this problem, we describe a robust yet simple method to make fluorescent amyloid fibrils through non-covalent incorporation of fluorophores into amyloid fibrils. We used aggregation protocol in which a small amount of fluorophore is incorporated into the amyloids, and this protocol does not alter the aggregation kinetics and the characteristic {beta}-sheet-conformers of the generated amyloid fibrils. We have successfully prepared fluorescent amyloid fibrils of Insulin, Lysozyme and A{beta}1-42, and the noncovalently incorporated fluorophores remained intact in the amyloid fibrils without leaching, even after serial-dilutions and prolonged-storage. Further, this method enables successful monitoring of cellular-internalization of the fluorescent amyloids into SH-SY5Y and A549 cells, and it also detects FRET-signals during interfibrillar interactions. The findings establish a simple and affordable protocol to prepare fluorescent amyloid structures, which may significantly help amyloid researchers working on both in vitro and animal model systems.

biophysics↗

bioRxiv2021

Computational and biochemical analyses reveal that cofilin-2 self assembles into amyloid-like structures and promotes the aggregation of other proteinaceous species: Pathogenic relevance to myopathies

Cofilin-2 is a member of the ADF/cofilin family, expressed extensively in adult muscle cells and involved in muscle maintenance and regeneration. Phosphorylated cofilin-2 is found in pre-fibrillar aggregates formed during idiopathic dilated cardiomyopathy. A recent study shows that phosphorylated cofilin-2, under oxidative distress, forms fibrillar aggregates. However, it remains unknown if cofilin-2 holds an innate propensity to form amyloid-like structures. In the present study, we employed various computational and biochemical techniques to explore the amyloid-forming potential of cofilin-2. We report that cofilin-2 possesses aggregation-prone regions (APRs), and these APRs get exposed to the surface, become solvent-accessible, and are involved in the intermolecular interactions during dimerization, an early stage of aggregation. Furthermore, the cofilin-2 amyloids, formed under physiological conditions, are capable of cross-seeding other monomeric globular proteins and amino acids, thus promoting their aggregation. We further show that Cys-39 and Cys-80 are critical in maintaining the thermodynamic stability of cofilin-2. The destabilizing effect of oxidation at Cys-39 but not that at Cys-80 is mitigated by Ser-3 phosphorylation. Cysteine oxidation leads to partial unfolding and loss of structure, suggesting that cysteine oxidation further induces early events of cofilin-2 aggregation. Overall, our results pose a possibility that cofilin-2 amyloidogenesis might be involved in the pathophysiology of diseases, such as myopathies. We propose that the exposure of APRs to the surface could provide mechanistic insight into the higher-order aggregation and amyloidogenesis of cofilin-2. Moreover, the cross-seeding activity of cofilin-2 amyloids hints towards its involvement in the hetero-aggregation in various amyloid-linked diseases.

biochemistry↗