Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly

Maria Bakou,Kathleen Hille,Michael Kracklauer,Anna Spanopoulou,Christina V Frost,Eleni Malideli,Li-Mei Yan,Andrea Caporale,Martin Zacharias,Aphrodite Kapurniotu

doi:10.1074/jbc.m117.774893

Maria Bakou, Kathleen Hille + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m117.774893

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Abstract

The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-β (Aβ) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aβ40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aβ40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aβ40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer's disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions.

Highlights

The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer’s disease amyloid-␤ (A␤) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases
The identified similarities and major differences controlling IAPP cross-peptide interaction with A␤40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer’s disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions
We (a) identified the aromatic/hydrophobic residues Phe[23] and Ile[26] within the well-known IAPP self-assembly–mediating segment FGAIL as hot spots of the IAPP interaction with A␤40 but not with IAPP (14, 15, 32, 37); (b) showed that Phe[15] or Phe[23] is required for IAPP interaction with A␤42 but not with IAPP; and (c) showed that the four aromatic/hydrophobic residues Phe[15], Leu[16], Phe[23], and Ile[26] within the IAPP amyloid core IAPP(15–29) act in concert to function as key molecular determinants of both IAPP self-assembly and its hetero-assembly with A␤40(42)

Summary

Results

We asked which are the key residues of the interaction of the partial IAPP “hot segments” IAPP(8 –18) and IAPP(22– 28) with IAPP or A␤40. In the case of the hetero-assemblies of A␤42 with double/triple Ala mutants, a strong destabilization (⌬⌬G Ͼ 2) was found only for the A15,23-A␤42 hetero-assemblies (Fig. 3, supplemental Fig. S1, and Tables 2 and 3) These latter results suggested that the presence of either Phe[15] or Phe[23] is required for the interaction of IAPP with A␤42. In strong contrast to IAPP-GI, the Ala-substituted IAPP-GI analogs were unable to block A␤40(42) and/or IAPP amyloidogenesis (supplemental Fig. S4) These results supported the suggestion that the four residues Phe[15], Leu[16], Phe[23], and Ile[26] play an important role in IAPP–A␤40(42) and IAPP–IAPP interactions. Self-assembly potentials (Kd(app) values) and factorial decrease of self-assembly potentials (Kd(mut)/Kd(wt)), increase of solubilities, and decrease of amyloidogenicities of mutants in comparison with IAPP

50–99 Ͻ100 Ͻ100 Ͻ100 Ͻ20 Ͻ20 Ͻ20 Ͻ20

Discussion

Experimental procedures