Beta Amyloid Oligomers with Higher Cytotoxicity have Higher Sidechain Dynamics.

The underlying biophysical principle governing the cytotoxicity of the oligomeric aggregates of ß-amyloid (Aß) peptides has long been an enigma. Here we show that the size of Aß40 oligomers can be actively controlled by incubating the peptides in reverse micelles. Our approach allowed for the first time a detailed comparison of the structures and dynamics of two Aß40 oligomers of different sizes, viz., 10 and 23 nm, by solid-state NMR. From the chemical shift data, we infer that the conformation and/or the chemical environments of the residues from K16 to K28 are different between the 10-nm and 23-nm oligomers. We find that the 10-nm oligomers are more cytotoxic, and the molecular motion of the sidechain of its charged residue K16 is more dynamic. Interestingly, the residue A21 exhibits unusually high structural rigidity. Our data raise an interesting possibility that the cytotoxicity of Aß40 oligomers could also be correlated to the motional dynamics of the sidechains.

Site specific NMR characterization of abeta-40 oligomers cross seeded by abeta-42 oligomers.

Extracellular accumulation of ß amyloid peptides of 40 (Aß40) and 42 residues (Aß42) has been considered as one of the hallmarks in the pathology of Alzheimer's disease. In this work, we are able to prepare oligomeric aggregates of Aß with uniform size and monomorphic structure. Our experimental design is to incubate Aß peptides in reverse micelles (RMs) so that the peptides could aggregate only through a single nucleation process and the size of the oligomers is confined by the physical dimension of the reverse micelles. The hence obtained Aß oligomers (AßOs) are 23 nm in diameter and they belong to the category of high molecular-weight (MW) oligomers. The solid-state NMR data revealed that Aß40Os adopt the structural motif of ß-loop-ß but the chemical shifts manifested that they may be structurally different from low-MW AßOs and mature fibrils. From the thioflavin-T results, we found that high-MW Aß42Os can accelerate the fibrillization of Aß40 monomers. Our protocol allows performing cross-seeding experiments among oligomeric species. By comparing the chemical shifts of Aß40Os cross seeded by Aß42Os and those of Aß40Os prepared in the absence of Aß42Os, we observed that the chemical states of E11, K16, and E22 were altered, whereas the backbone conformation of the ß-sheet region near the C-terminus was structurally invariant. The use of reverse micelles allows hitherto the most detailed characterization of the structural variability of Aß40Os.

Role of lysine residue of islet amyloid polypeptide in fibril formation, membrane binding, and inhibitor binding.

The aggregation of islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of type 2 diabetes (T2D). In T2D, this peptide aggregates to form amyloid fibrils; the mechanism responsible for islet amyloid formation is unclear. However, it is known that the aggregation propensity of IAPP is highly related to its primary sequence. Several residues have been suggested to be critical in modulating IAPP amyloid formation, but role of the sole lysine residue at position 1 (Lys-1) in IAPP has not been discussed. In our previous study, we found that glycated IAPP can form amyloid faster than normal IAPP and induce normal IAPP to expedite the aggregation process. To gain more insight into the contribution of Lys-1 in the kinetics of fibril formation, we synthesized another two IAPP variants, K1E-IAPP and K1Nle-IAPP, in which the Lys residue was mutated to glutamate and norleucine, respectively. Interestingly, we observed that the negative or neutral charged side chain at this position was preferred for amyloid formation. The findings suggested this residue may take part in the inter- or intra-molecular interaction during IAPP aggregation, even though it was proposed not to be in part of fibril core structure. Our data also revealed that the inhibitory mechanism of some inhibitors for IAPP aggregation require reaction with Lys-1. Modifications of Lys-1, such as protein glycation, may affect the effectiveness of the inhibitory action of some potential drugs in the treatment of amyloidosis.

Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide.

Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its ß-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.