Identification of key regions and residues controlling Aβ folding and assembly

www.nature.com/scientificreports OPEN Received: 24 May 2017 Accepted: 15 August 2017 Published: xx xx xxxx Identification of key regions and residues controlling Aβ folding and assembly Eric Y. Hayden1, Kimberly K. Hoi1,4, Jasmine Lopez1, Mohammed Inayathullah Margaret M. Condron1 & David B. Teplow 1,2 3 , Amyloid β-protein (Aβ) assembly is hypothesized to be a seminal neuropathologic event in Alzheimer’s disease (AD). We used an unbiased D-amino acid substitution strategy to determine structureassembly relationships of 76 different Aβ40 and Aβ42 peptides. We determined the effects of the substitutions on peptide oligomerization, secondary structure dynamics, fibril assembly dynamics, and fibril morphology. Our experiments revealed that the assembly of Aβ42 was more sensitive to chiral substitutions than was Aβ40 assembly. Substitutions at identical positions in the two peptides often, but not always, produced the same effects on assembly. Sites causing substantial effects in both Aβ40 and Aβ42 include His14, Gln15, Ala30, Ile31, Met35, and Val36. Sites whose effects were unique to Aβ40 include Lys16, Leu17, and Asn 27, whereas sites unique to Aβ42 include Phe20 and Ala21. These sites may be appropriate targets for therapeutic agents that inhibit or potentiate, respectively, these effects. Alzheimer’s disease (AD) is the most common cause of late-life dementia1 and recently has been identified as the 6th leading cause of death in the U.S.2. Thus, there is a compelling need for the development of approaches to prevent, ameliorate, or cure this tragic disorder. A predominant working hypothesis of disease causation posits that oligomeric forms of the amyloid β-protein (Aβ) are key neurotoxic agents3. If so, then therapeutic drug development requires appropriate targeting of these assemblies. A variety of targeting strategies have been executed, including those directed against the enzymes responsible for Aβ production (β-secretase and γ-secretase)4 or immunogenic sites on monomeric, oligomeric, and fibrillar forms of Aβ5. Unfortunately, none have resulted in an FDA-approved therapeutic agent6. Aβ exists in humans predominantly in two forms, Aβ40 and Aβ42, that are 40 or 42 amino acid residues in length, respectively4. Aβ42 appears to be the most disease-relevant peptide7–9. Mutations in the gene encoding the amyloid precursor protein (APP), from which Aβ42 is produced, lead to single amino acid substitutions linked to familial forms of AD, cerebral amyloid angiopathy (CAA), or AD with CAA. Most mutations result in single amino acid substitutions10–22. One results in the deletion of Glu2210. In vitro studies of the conformational dynamics and assembly of Aβ peptides containing these substitutions show that they facilitate folding, oligomerization, or fibril formation by the initially disordered Aβ monomer (for reviews, see refs23–25). However, familial AD (with or without CAA) is estimated to account for <1% of all AD cases26. This means that in the majority of AD cases, Aβ-mediated neurotoxicity and plaque formation are caused by wild type Aβ. We sought here to determine which amino acids in wild type Aβ40 and Aβ42 have the greatest effects on peptide folding and assembly. Prior approaches for answering this question often have relied on amino acid substitution strategies, which have been informative and useful. However, by definition, the substituted amino acids differ from the wild type amino acids in polarity, charge, hydrophobicity, or size of the amino acid side-chains. These differences per se may be responsible for any changes, or lack of changes, in peptide folding and assembly, as opposed to the differences indicating how the wild type amino is involved these processes. To avoid these interpretive difficulties, we employed a scanning D-amino acid substitution strategy. We did so because chiral substitution only affects the orientation of the side-chain relative to the peptide backbone27–30. These substitutions thus reveal amino acids whose side-chains are involved in inter-atomic interactions that are exquisitely sensitive 1 Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA. 2Molecular Biology Institute and Brain Research Institute; UCLA, Los Angeles, CA, 90095, USA. 3Biomaterials and Advanced Drug Delivery Laboratory, School of Medicine, Stanford University, Palo Alto, CA, 94304, USA. 4Present address: Department of Pediatrics and Department of Neurology, UCSF, San Francisco, CA, 94158, USA. Correspondence and requests for materials should be addressed to D.B.T. (email: ) Scientific Reports | 7: 12434 | DOI:10.1038/s41598-017-10845-6 1 www.nature.com/scientificreports/ to structural perturbation and thus may be of special importance in controlling Aβ assembly and toxicity. In much the same way that study of transition states in chemical and enzymatic reactions are critical for establishing a mechanistic understanding of such reactions, chiral inversions may enable the study of peptide folding trajectories rarely traversed by the wild type peptide but that are critical for the production of conformers associated with pathologic Aβ assembly31,32 (see Raskatov and Teplow30 for a theoretical treatment of the scanning D-amino acid strategy). Our experimental design comprised initial studies of the effects of scanning di-D amino acid substitution on peptide oligomerization and fibril formation, followed by determination of the effects of single D-amino acid substitutions chosen based on the data obtained in the initial studies. This allowed us to determine the effects of specific amino acids on peptide oligomerization, secondary structure, fibril assembly, and fibril morphology and if any differences in effects were observed between Aβ40 and Aβ42. Materials and Methods Peptide synthesis and preparation. Aβ40, Aβ42, di-D-amino acid substituted peptides, and single D-amino acid substituted peptides were synthesized, purified, and characterized in the Biopolymer Lab at UCLA33. Briefly, peptide synthesis was performed on an automated peptide synthesizer (Model 433 A, Applied Biosystems, Foster City, CA) using 9-fluorenylmethoxycarbonyl-based methods at a 0.25 mmol scale. Peptides were purified using reverse phase high-performance liquid chromatography (RP-HPLC). The purity of the peptides were >95%. Quantitative amino acid analysis and mass spectrometry yielded the expected compositions and molecular weights, respectively, for each peptide. Purified peptides were stored as lyophilizates at −20 °C. Peptide lyophilizates were solvated initially in 10% (v/v) 60 mM NaOH, 45% (v/v) H2O (prepared using a Synthesis A10 water purification system (Millipore, Bedford, MA)), and 45% (v/v) 22.2 mM sodium phosphate, pH 7.4, on ice. The solutions were sonicated for 1 min in an ultrasonic water bath (Model 1510, Branson Ultrasonics Corp., Danbury, CT) and then they were filtered using a Microcon centrifugal filter (30 kDa molecula (...truncated)