Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin

Abstract

Background: Insulin is a model amyloidogenic protein.Results: Limited proteolysis of bovine insulin dimers with pepsin releases highly fibrillation-prone two-chain fragments. Conclusion: Dynamics of the disulfide-bonded N-terminal fragments of A-and B-chains may strongly contribute to insulin amyloidogenesis. Significance: Highly aggregation-prone regions of protein molecules may be revealed by partial proteolysis of the native state.

Beyond amino acid sequence: disulfide bonds and the origins of the extreme amyloidogenic properties of insulin's H‐fragment

“…In comparison to intact bovine insulin (BI), aggregation of either peptide is strongly accelerated. The transition is particularly fast for synthetic H-fragment, as it takes place without measurable lag phase which is in accordance with the previously reported behavior of H-fragment samples obtained through aggregation-quenched partial proteolysis of insulin with pepsin [31]. Aggregation of H-fragment monomers is complete within the first 3 h. On the other hand, de novo fibrillization of AB-fragment is significantly slower: after the approximately 6-h-long lag phase, the elongation phase sets in reaching plateau after the following 4 h. The visible difference between final ThT fluorescence intensity levels for H and AB aggregation cannot be attributed to a lesser amount of [AB] fibrils as the starting concentrations of H and AB were identical while negligible fractions of monomers remained in solution at the end of aggregation.…”

“…A may be explained by the presence of in situ released reduced A‐CC fragment undergoing fibrillization on its own (somehow decelerated due to Cys6A‐Cys11A bond reduction) with the reduced non‐amyloidogenic B‐fragment remaining in solution. Only half of the overall peptide content (in terms of mass) participates in the fibrillization under these conditions which contributes to the visible reduction of ThT fluorescence intensity also observed when enzymatically derived H‐fragment was made to reassociate in the presence of TCEP (). In the case of AB, reduction of the single Cys7A‐Cys7B bond can only enhance aggregation by untethering the amyloidogenic A‐fragment from the aggregation‐resistant B‐fragment.…”

“…During aggregation of H‐fragments, the otherwise non‐amyloidogenic B‐chain fragment appears to be ‘dragged along’ through the Cys7A‐Cys7B tether and forced to attain the β‐sheet conformation (remarkably, infrared spectra of [BI], [H], and [ACC] fibrils indicate high and similar β‐sheet contents (Figs and ). We have shown previously () that the Tango algorithm fails to predict the scale of amyloidogenic potential of fully reduced A‐chain part of the H‐fragment. In most cases, topological constrains from disulfide bridges cannot be even taken into account by such computational methods.…”

Mellitate: A multivalent anion with extreme charge density causes rapid aggregation and misfolding of wild type lysozyme at neutral pH

“…Amorphous protein aggregates are expected to exhibit lower stability than fibrils (e.g. [ 30 – 31 ]). Such HEWL-MA aggregates are likely to trap ThT causing a moderate increase in quantum yield of its fluorescence following from a partial restriction of dye’s intramolecular rotational freedom which would be entirely unrelated to and independent of presence of amyloid fibrils.…”

The Kinetics of Amyloid Fibril Formation by de Novo Protein Albebetin and Its Mutant Variants

“…However, it should be mentioned that the role of disulfide bond in amyloid fibril formation is not always interpreted using the paradigm of native state stabilization. Thus, it has been demonstrated for the highly amyloidogenic two-chain H-fragment of insulin that the presence of a disulfide bond plays a decisive role for its amyloidogenicity: association of H-fragment monomers to fibrils decreased abruptly in the presence of an agent reducing disulfide bonds [33]. In a similar The disulfide bond in albebetin can be formed between Cys residues at positions 8 and 43, which are symmetrically located in identical structural elements of the ABB molecule (as it is in the case for His30 and His65 residues).…”