Structural studies and cytotoxicity assays of “aggregation‐prone” IAPP_8–16 and its non‐amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin

Abstract

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic β-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8-20, 20-29, and 30-37. A smaller fragment of the 8-20 region, spanning residues 8-16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8-16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8-16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8-16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8-16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8-16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

Residue specific effects of human islet polypeptide amyloid on self-assembly and on cell toxicity

“…Later, a number of mutations within residue 20 (Ser/Gly, Ser/Lys) were investigated and the results highlighted the importance of this residue in hIAPP structure and fibril formation [35e37]. In addition, multiple substitutions outside of the 20e29 region also led to nonamyloidogenic or less amyloidogenic peptides suggesting the importance of others regions such as 8e16, 13e19 and 31e37 [8,15,38,39]. Our study, revealing that histidine18 is important on allowing fibrillation to proceed, is consistent with a previous study that demonstrates that the mutation R18H is sufficient to render mIAPP amyloidogenic [19].…”

Heterotypic interactions in amyloid function and disease

“…Atomic structures of APR segment homologs from major prion protein (PrP) highlighted that sequence specificity critically confers interspecies transmissibility barriers [146]. Rationally designed single-position APR variants can alter both the aggregation propensity and cytotoxicity of hIAPP [147,148], with additional work supporting that sequence variants can disrupt self-association and suppress aggregation of other proteins [149,150]. Co-expression studies of unrelated aggregating peptides, sharing no sequence similarity, resulted to the formation of separate inclusion bodies in mammalian, as well as E. coli cells [151].…”

A common ‘aggregation‐prone’ interface possibly participates in the self‐assembly of human zona pellucida proteins

“…Nevertheless, organisms spanning from bacteria to humans express proteins that natively form filamentous arrangements, known as functional amyloid, sharing common features and qualities of amyloid, in order to support fundamental biological processes . Experimental data has highlighted that the overall self‐aggregation tendency of a protein may be directed by the presence of short sequence stretches with an inherent aggregation propensity . Recently, peptide segments corresponding to a possible ‘aggregation‐prone’ interaction site, composed of the A and G β‐strands of the putative ZP‐N domain from hZP1, therein and henceforth called the AG interface, were shown to possess characteristic amyloidogenic properties .…”