Terminal Capping of an Amyloidogenic Tau Fragment Modulates Its Fibrillation Propensity.

Aberrant protein folding leading to the formation of characteristic cross-ß-sheet-rich amyloid structures is well known for its association with a variety of debilitating human diseases. Often, depending upon amino acid composition, only a small segment of a large protein participates in amyloid formation and is in fact capable of self-assembling into amyloid, independent of the rest of the protein. Therefore, such peptide fragments serve as useful model systems for understanding the process of amyloid formation. An important factor that has often been overlooked while using peptides to mimic full-length protein is the charge on the termini of these peptides. Here, we show the influence of terminal charges on the aggregation of an amyloidogenic peptide from microtubule-associated protein Tau, implicated in Alzheimer's disease and tauopathies. We found that modification of terminal charges by capping the peptide at one or both of the termini drastically modulates the fibrillation of the hexapeptide sequence paired helical filament 6 (PHF6) from repeat 3 of Tau, both with and without heparin. Without heparin, the PHF6 peptide capped at both termini and PHF6 capped only at the N-terminus self-assembled to form amyloid fibrils. With heparin, all capping variants of PHF6, except for PHF6 with both termini free, formed typical amyloid fibrils. However, the rate and extent of aggregation both with and without heparin as well as the morphology of aggregates were found to be highly dependent on the terminal charges. Our molecular dynamics simulations on PHF6 capping variants corroborated our experiments and provided critical insights into the mechanism of PHF6 self-assembly. Overall, our results emphasize the importance of terminal modifications in fibrillation of small peptide fragments and provide significant insights into the aggregation of a small Tau fragment, which is considered essential for Tau filament assembly.

Catalytic Prion-Like Cross-Talk between a Key Alzheimer's Disease Tau-Fragment R3 and the Type 2 Diabetes Peptide IAPP.

The aberrant association of proteins/peptides is implicated in the etiology and pathogenesis of a variety of human diseases. In general, the primary protein component responsible for the formation of aggregates is different in each case and is specific to a particular disease condition. However, there are instances where multiple protein aggregates have been found to coexist in the same or different tissue(s), thereby leading to mixed pathologies and exacerbation of disease symptoms. In this context, a strong link has been established between Alzheimer's disease (AD) and type 2 diabetes (T2D). However, the underlying molecular details still remain elusive. Here, we report the direct interaction of an AD-associated amyloidogenic cytotoxic fragment of Tau (R3:306-336) with islet amyloid polypeptide (IAPP) implicated in T2D. Using ion-mobility mass spectrometry (IM-MS) in conjunction with fluorescence spectroscopy, circular dichroism, and transmission electron microscopy, we have been able to provide critical mechanistic insights into these interactions. Our IM-MS data showed the formation of hetero-oligomers of R3 and IAPP. Additionally, using IM-MS, we found that the amyloidogenic extended beta hairpin conformation of IAPP is favored much more in the R3-IAPP mixture, when compared with IAPP alone. Furthermore, we found that the oligomerization of R3 occurs much faster in the presence of IAPP. We also observed a secondary nucleation step in our kinetics data for the R3-IAPP mixture. We believe that the secondary nucleation step is demonstrative of R3 aggregation which otherwise requires the presence of anionic cofactors. Our results provide the first experimental evidence for direct molecular interaction between Tau and IAPP and highlights the repercussion of possible "prion-like" cross-talk in the proliferation of diseases that are associated with different tissues/organs.

Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide ß-Hairpin.

Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Coaggregation of heterogeneous peptide species can occur, however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP106-126) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of nonhomologous protein assembly as they coaggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only forms pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic ß-hairpin conformation. Further, when PrP106-126 was combined with non-amyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing ß-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic ß-hairpin structure, it did so in the presence of PrP106-126 and underwent a conformational transition to ß-sheet in solution. We also find that PrP106-126 forms hetero-oligomers with the IAPP8-20 fragment but not with the "aggregation hot spot" IAPP20-29 fragment. PrP106-126 apparently induces IAPP into a ß-hairpin structure within the PrP:IAPP heterodimer complex and then, through ligand exchange, catalytically creates the amyloidogenic ß-hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model that provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.