The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Abstract

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12-18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Fibril formation and toxicity of the non-amyloidogenic rat amylin peptide

“…Previous studies have demonstrated that human amylin fibrils show interactions with Congo red (Lim et al, 2008;Milton and Harris, 2010;Milardi et al, 2011). Our observations for Congo red interactions with A␤ indicate that the interaction can also occur in the absence of fibrils by TEM (Milton and Harris, 2009).…”

JC virus agnoprotein enhances large T antigen binding to the origin of viral DNA replication: Evidence for its involvement in viral DNA replication

“…Importantly, Phe residues, in general, are known to play diverse but important roles in the function of many different proteins through hydrophobic interactions and possibly through “cation-π” and “anion-π” interactions (Gallivan and Dougherty, 1999; Jackson et al, 2007; Philip et al, 2011; Pless et al, 2008; Pletneva et al, 2001; Shi et al, 2002). Such interactions mediate protein-protein interactions at protein interfaces and also aid to the folding and stability of many different proteins (Bowden et al, 2008; Gallivan and Dougherty, 1999; Jackson et al, 2007; King et al, 2011; Milardi et al, 2011; Philip et al, 2011; Pless et al, 2008; Pletneva et al, 2001; Shi et al, 2002). As such, it is conceivable that the Phe residues of agnoprotein may participate in such interactions to form homodimers between its monomers or heterodimers between agnoprotein and other cellular or viral proteins and thereby contribute to the function of agnoprotein during the viral replication cycle.…”

“…Phenylalanine residues are known to play important roles in functions of many different proteins, including in mediation of protein-protein interactions, protein folding and stability (Bowden et al, 2008; Gallivan and Dougherty, 1999; King et al, 2011; Milardi et al, 2011; Pless et al, 2008; Pletneva et al, 2001; Shi et al, 2002). Phe residues accomplish such functions through hydrophobic interactions mediated by the π–π stacking of the aromatic ring of Phe or “cation-π” interactions (Bowden et al, 2008; Gallivan and Dougherty, 1999; King et al, 2011; Milardi et al, 2011; Pless et al, 2008; Pletneva et al, 2001; Shi, Olson, and Kallenbach, 2002) or “anion-π interactions (Jackson et al, 2007; Philip et al, 2011) or through the combination of all these three types of interactions.…”

“…Phenylalanine residues have been reported to play diverse but important regulatory roles in functions of many different proteins through hydrophobic interactions [pi (π)-pi(π) stacking] (Bowden et al, 2008; Brinda, Kannan, and Vishveshwara, 2002; Dhe-Paganon et al, 2004; King et al, 2011; Milardi et al, 2011). These residues were also found to be involved in “cation-π” interactions which could take place between Phe and charged residues (Arg or Lys or His) (Gallivan and Dougherty, 1999; Pless et al, 2008; Pletneva et al, 2001; Shi et al, 2002).…”

Aromaticity and amyloid formation: Effect of π-electron distribution and aryl substituent geometry on the self-assembly of peptides derived from hIAPP22–29

“…Raleigh and co-workers have demonstrated that aromatic amino acids are not an absolute requirement for amyloid formation by both full-length and peptide fragments derived from hIAPP. Replacement of Phe-15 and 23 in the peptide fragments hIAPP 12–19 (LANVFLVH) and hIAPP 22–29 (NFGAILSS) respectively with leucine resulted in peptides still capable of forming amyloid [34,35]. Likewise, substitution of all three aromatic amino acids in full-length hIAPP with Leu did not inhibit the polypeptide from forming aggregates [36].…”