Amyloid-α Peptide Formed through Alternative Processing of the Amyloid Precursor Protein Attenuates Alzheimer's Amyloid-ß Toxicity via Cross-Chaperoning.

Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-ß (Aß), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aß, aggregates rapidly to form amyloids and can expedite the aggregation of Aß through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aß to form amyloid aggregates that are less toxic than pure Aß aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aß in solution. This is the first evidence that Aα can coassemble with Aß and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.

A robust preparation method for the amyloidogenic and intrinsically disordered amyloid-α peptide.

Recent findings suggest that amyloid-ß (Aß) may not be the only peptidic culprit for the cognitive decline observed in patients with Alzheimer's disease. A C-terminal fragment of Aß, amyloid-α (Aα), also known as p3, has been shown to form amyloidogenic oligomers and fibrils more rapidly than Aß. However, the insolubility and aggregation propensity of this 24-26-residue peptide make it exceptionally difficult to produce, purify, and subsequently study. This paper reports a reproducible, multi-step method for the purification and pre-treatment of Aα and related analogues, yielding 95%-99% pure peptides. We anticipate that the methods described herein will permit previously inaccessible biophysical and biological experiments that may be critical to understanding the role of this too long overlooked peptide in AD disease pathology.

A crystal-structural study of Pauling-Corey rippled sheets.

Following the seminal theoretical work on the pleated ß-sheet published by Pauling and Corey in 1951, the rippled ß-sheet was hypothesized by the same authors in 1953. In the pleated ß-sheet the interacting ß-strands have the same chirality, whereas in the rippled ß-sheet the interacting ß-strands are mirror-images. Unlike with the pleated ß-sheet that is now common textbook knowledge, the rippled ß-sheet has been much slower to evolve. Much of the experimental work on rippled sheets came from groups that study aggregating racemic peptide systems over the course of the past decade. This includes MAX1/DMAX hydrogels (Schneider), L/D-KFE8 aggregating systems (Nilsson), and racemic Amyloid ß mixtures (Raskatov). Whether a racemic peptide mixture is "ripple-genic" (i.e., whether it forms a rippled sheet) or "pleat-genic" (i.e., whether it forms a pleated sheet) is likely governed by a complex interplay of thermodynamic and kinetic effects. Structural insights into rippled sheets remain limited to only a very few studies that combined sparse experimental structural constraints with molecular modeling. Crystal structures of rippled sheets are needed so we can rationally design rippled sheet architectures. Here we report a high-resolution crystal structure, in which (l,l,l)-triphenylalanine and (d,d,d)-triphenylalanine form dimeric antiparallel rippled sheets, which pack into herringbone layer structures. The arrangements of the tripeptides and their mirror-images in the individual dimers were in excellent agreement with the theoretical predictions by Pauling and Corey. A subsequent mining of the PDB identified three orphaned rippled sheets among racemic protein crystal structures.

Alzheimer's Disease "Non-amyloidogenic" p3 Peptide Revisited: A Case for Amyloid-α.

Amyloid-ß (Aß) is an intrinsically disordered peptide thought to play an important role in Alzheimer's disease (AD). It has been the target of most AD therapeutic efforts, which have repeatedly failed in clinical trials. A more predominant peptidic fragment, formed through alternative processing of the amyloid precursor protein, is the p3 peptide. p3 has received little attention, which is possibly due to the prevailing view in the AD field that it is "non-amyloidogenic." By probing the self-assembly of this peptide, we found that p3 aggregates to form oligomers and fibrils and, when compared with Aß, displays enhanced aggregation rates. Our findings highlight the solubilizing effect of the N-terminus of Aß and the favorable formation of structures formed through C-terminal hydrophobic peptide interfaces. Based on our findings, we suggest a reevaluation of the current therapeutic approaches targeting only the ß-secretase pathway of AD, given that the α- secretase pathway is also amyloidogenic.

Is the p3 Peptide (Aß17-40, Aß17-42) Relevant to the Pathology of Alzheimer's Disease?1.

Despite the vast heterogeneity of amyloid plaques isolated from the brains of those with Alzheimer's Disease (AD), the basis of the Amyloid Cascade Hypothesis targets a single peptide, the amyloid-ß (Aß) peptide. The countless therapeutic efforts targeting the production and aggregation of this specific peptide have been met with disappointment, leaving many to question the role of Aß in AD. An alternative cleavage product of the Amyloid-ß protein precursor, called the p3 peptide, which has also been isolated from the brains of AD patients, has been largely absent from most Aß-related studies. Typically referred to as non-amyloidogenic and even suggested as neuroprotective, the p3 peptide has garnered little attention aside from some conflicting findings on cytotoxicity and potential self-assembly to form higher order aggregates. Herein, we report an extensive analysis of the findings surrounding p3 and offer some evidence as to why it may not be as innocuous as previously suggested.

A Facile Method for the Separation of Methionine Sulfoxide Diastereomers, Structural Assignment, and DFT Analysis.

Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO2 chromatography and allows obtaining both diastereomers in purities exceeding 99 %. 1 H NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2 kcal mol-1 , highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.

Using mirror-image peptides to enhance robustness and reproducibility in studying the amyloid ß-protein.

Alzheimer's disease, the most common form of dementia, is a devastating disease that affects over 44 million people worldwide. One etiological agent of Alzheimer's, the amyloid ß-protein (Aß), is an aggregation-prone, intrinsically disordered peptide that can form a wide variety of aggregates. The pathways by which Aß aggregates in order to exert its toxicity, referred to as the Amyloid Cascade, remains largely elusive despite substantial deconvolution efforts. Preparing high-quality material that exhibits reproducible biophysical characteristics has proven challenging. Herein, we propose that mirror-image peptides can be used to rigorously control Aß preparation quality.

Chirality Dependence of Amyloidâ€ ß Cellular Uptake and a New Mechanistic Perspective.

Amyloidâ€…ß is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloidâ€…ß and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.

Hydrogen Shifts in Aryl Radicals and Diradicals: The Role of m-Benzynes.

Density functional and coupled cluster results are presented for hydrogen shifts in radicals derived from polycyclic aromatic hydrocarbons (PAHs) and for rearrangement mechanisms for several phenylenes. RCCSD(T)/cc-pVDZ//UBLYP/cc-pVDZ free energy barriers for 1,4-H shifts at 298 K are consistently predicted to be ca. 25 kcal/mol, whereas barriers for 1,5- and 1,6-shifts range from 6 to 28 kcal/mol. The barriers correlate reasonably well with the distance from the radical center to the shifting hydrogen in the reactant. Proposed mechanisms (via diradical intermediates) of known rearrangements of linear [3]phenylene, benzo[b]biphenylene, and angular [4]phenylene have BD(T)/cc-pVDZ//(U)BLYP/cc-pVDZ computed barriers of 74-82 kcal/mol, consistent with pyrolysis temperatures of 900 to 1100 °C. Hydrogen shift reactions in most of the aryl diradicals arising from phenylenes produce m-benzyne intermediates which, despite being 8-15 kcal/mol more stable than other diradicals involved in the pathways, do not significantly lower the computed overall free energies of activation.