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To date, most research on amyloid aggregation has focused on describing the structure of amyloids and the kinetics of their formation, while the conformational stability of fibrils remains insufficiently explored. The aim of this work was to investigate the effect of amino acid substitutions on the stability of apomyoglobin (ApoMb) amyloids. A study of the amyloid unfolding of ApoMb and its six mutant variants by urea has been carried out. Changes in the structural features of aggregates during unfolding were recorded by far-UV CD and native electrophoresis. It was shown that during the initial stage of denaturation, amyloids' secondary structure partially unfolds. Then, the fibrils undergo dissociation and form intermediate aggregates weighing approximately 1 MDa, which at the last stage of unfolding decompose into 18 kDa monomeric unfolded molecules. The results of unfolding transitions suggest that the stability of the studied amyloids relative to the intermediate aggregates and of the latter relative to unfolded monomers is higher for ApoMb variants with substitutions that increase the hydrophobicity of the residues. The results presented provide a new insight into the mechanism of stabilization of protein aggregates and can serve as a base for further investigations of the amyloids' stability. [ABSTRACT FROM AUTHOR]

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A). [ABSTRACT FROM AUTHOR]

Cytotoxicity of amyloid fibrils has been shown to depend on their structure. However, specific features of toxic and non-toxic amyloids remain unclear. Here we focus on the relationship between structural characteristics of the fibrils and their cytotoxicity. Bovine carbonic anhydrase B (BCAB) serves as the object of this study because its amyloids reduce cell viability. Limited proteolysis and mass spectrometry were used to determine BCAB regions forming the core of amyloid fibrils. Four BCAB mutants with substitutions reducing hydrophobicity in the regions important for amyloid formation were obtained to study the kinetics of aggregation, structural features, and cytotoxicity of the amyloids. We demonstrate that fibrils of WT BCAB, L78A, L139A, and M239A variants display a pronounced toxic effect on eukaryotic cells, while I208A mutation significantly reduces the cell-damaging effect of amyloids. The data obtained conclude that cytotoxicity of BCAB fibrils does not depend on their length, secondary structure, and exposure of hydrophobic groups to the solvent. A distinctive feature of the low-toxic I208A fibrils is their specific morphology characterized by the lateral protofilaments association and formation of fibril-ribbons. • Amyloid fibrils of BCAB and its mutants decrease eukaryotic cells viability. • The fibrils cytotoxicity doesn't depend on their length, secondary structure, and ANS binding. • Specific feature of the low-toxic amyloids is the lateral protofilaments association. [ABSTRACT FROM AUTHOR]

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins' aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions. [ABSTRACT FROM AUTHOR]

In most cases high cytotoxicity is characteristic of aggregates formed during lag phase of amyloid formation, whereas mature fibrils represent the depot of protein molecules incapable of damaging cell membranes. However, new experimental data show that in cases of some proteins the fibrils are the most toxic type of aggregates. Meanwhile, structural characteristics of cytotoxic fibrils and mechanisms of their cell damaging action are insufficiently explored. This work is dedicated to studying amyloid aggregation of bovine carbonic anhydrase (BCA) and effect of aggregates formed at different stages of amyloid formation on viability of the cells. Here we demonstrate that oligomers formed during lag phase do not decrease cell viability, whereas protofibrils and amyloids of BCA are cytotoxic. Obtained results allow concluding that toxicity of BCA aggregates is associated with the presence of amyloid cross-β-structure, which signature is absorbance peak at low wavenumbers at FTIR spectra (1615-1630 cm−1). Our data suppose that cross-β-core of ВСА amyloid fibrils is responsible for their cytotoxicity. • Oligomers formed during lag phase of bovine carbonic anhydrase (BCA) amyloid aggregation do not decrease cell viability. • Protofibrils and mature fibrils of BCA are cytotoxic. • Toxicity of BCA aggregates is associated with the presence of amyloid cross-β-structure. [ABSTRACT FROM AUTHOR]