Analysis of the Amyloidogenic Potential of Pufferfish (Takifugu rubripes) Islet Amyloid Polypeptide Highlights the Limitations of Thioflavin-T Assays and the Difficulties in Defining Amyloidogenicity

Abstract

Islet amyloid polypeptide (IAPP, amylin) forms pancreatic amyloid in type-2 diabetes, a process that contributes to the loss of β-cell mass in the disease. IAPP has been found in all higher organisms examined, but not all species form amyloid and the ability to do so correlates with the primary sequence. The amyloidogenic potential of fish IAPPs have not been examined, although fish have been proposed as a source for xenobiotic transplantation. The sequence of pufferfish IAPP (Takifugu rubripes) is known and is the most divergent from human IAPP of any reported IAPP sequence, differing at eleven positions including seven located within residues 20 to 29, a segment of the molecule that is important for controlling amyloidogenicity. Several of the substitutions found in pufferfish IAPP are non-conservative including Ser to Pro, Asn to Thr, Ala to Tyr and Leu to Tyr replacements and several of these have not been reported in mammalian IAPP sequences. Amyloid prediction programs give conflicting results for pufferfish IAPP. CD spectroscopy, FTIR, and transmission electron microscopy reveal that pufferfish IAPP forms amyloid and does so more rapidly than human IAPP in tris buffer at pH 7.4, but does so more slowly in phosphate buffered saline (PBS) at pH 7.4. Molecular dynamics simulations indicate that the pufferfish sequence is compatible with models of IAPP amyloid. The fish polypeptide does not significantly bind to thioflavin-T in tris and does so only weakly in PBS. The results highlight difficulties with thioflavin-T assays and the ambiguity in defining amyloidogenicity.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

“…4C). Although seemingly contradictory, it has been demonstrated previously that amyloids can possess ␤-strand content and be unable to bind ThT (30). Moreover, the TEM images of the preformed fibers treated with DLPC displayed a morphology distinct from mature fibers formed in solution (Figs.…”

“…When A␤ aggregated in the presence of DLPC at stoichiometric (10 M; 1:1 lipid/peptide ratio) or higher lipid concentrations, ThT fluorescence remained at baseline levels, which suggests no fibril formation. ThT will occasionally provide false positives of inhibition resulting from dye displacement or the inability of unique amyloid sequences to bind the dye, despite containing the canonical ␤-strand fold (29,30). To confirm that the reduction in ThT fluorescence was the result of structural change, CD and TEM were performed on end stage aggregates (Fig.…”

Islet Amyloid Polypeptide Membrane Interactions: Effects of Membrane Composition

“…Transmission electron microscopy (TEM) was used to visualize the final products of the amyloid formation assays to provide an independent test of amyloid formation. This is an important control since thioflavin-T is an extrinsic probe and sometimes gives a low signal even in the presence of amyloid fibrils 71 . As expected, and in agreement with earlier studies, the rate of hIAPP amyloid formation was notably enhanced in the presence of vesicles containing high percentages of anionic lipids (Figure-2) 42, 43, 49, 52, 57 .…”

Evolutionary Adaptation and Amyloid Formation: Does the Reduced Amyloidogenicity and Cytotoxicity of Ursine Amylin Contribute to the Metabolic Adaption of Bears and Polar Bears?

“…It is clearly important to experimentally test the two polypeptides, however, since the methods fail to predict that certain regions of human amylin are amyloidogenic, even though they are part of the core cross‐β structure in all existing structural models human amylin amyloid fibrils. In addition, these prediction programs give inconsistent results for other variants of amylin, and relative amyloidogenicity can vary, depending on the choice of buffer …”

“…Amyloid formation by amylin and other amyloidogenic proteins is a multiphasic process, composed of a lag phase, in which little or no detectable amyloid fibrils are formed; followed by a growth phase, where fibrils lengthen; and a final plateau phase, in which amyloid fibrils exist in equilibrium with soluble peptide (Figure S2). ThioflavinT is a small dye that is widely used in biophysical studies of amyloid formation ,. The dye is weakly fluorescent in the buffer and does not bind to preamyloid intermediates, but does bind to amyloid fibrils and experiences an increase in quantum yield when it does so.…”