Stabilization, Assembly, and Toxicity of Trimers Derived from Aβ.docx

This is an open access article published under an ACS AuthorChoice License, which permitscopying and redistribution of the article or any adaptations for non-commercial purposes.ArticleTIAICISJOURNAL OF THE AMERICAN CHEMICAL SOCIETYpubs.acs.org/JACSStabilization, Assembly, and Toxicity of Trimers Derived from ApAdam G. Kreutzer, Stan Yoo, Ryan K. Spencer, and James S. Nowick*GDepartment of Chemistry, University of California, Irvine, Irvine, California 92697-2025, United States* Supporting InformationABSTRACT: Oligomers of the P-amyloid peptide A(3 haveemerged as important contributors to ncurodcgcncration inAlzheimer's disease. Mounting evidence suggests that Aptrimers and higher-order oligomers derived from trimershave special significance in the early stages of Alzheimer'sdisease. Elucidating the structures of these trimers andhigher-order oligomers is paramount for understandingtheir role in neurodegeneration. This paper describes thedesign, synthesis,X-ray crystallographic structures, and biophysical and biological properties of two stabilized trimers derived from thecentral and C-terminal regions of Ap. These triangular trimers are stabilized through three disulfide cross-links betweenthe monomer subunits, The X-ray crystallographic structures reveal that the stabilized trimers assemble hierarchically toR)rm hexamers, dodccamcrs, and annular porclikc structures. Solution-phase biophysical studies reveal that the stabilizedtrimers assemble in solution to form oligomers that recapitulate some of the higher-order assemblies observedcrystallographically. The stabilized trimers share many of the biological characteristics of oligomers of full-length Ap,including toxicity toward a neuronally derived human cell line, activation of caspase-3 mediated apoptosis, and reactivitywith the oligomer-specific antibody All. These studies support the biological significance of the triangular trimer assemblyoTTPP-hairpins and may offer a deeper understanding of thessonJB POL-S 二 qndoJeqs A3EE 三号 oi MOL- uo suo 二 do sa>.£op388u-Bqs/8JOsoBsqnd/_sd=q UssuHe9N9ZN 一急 ZZOZ Nz 工 co com. w 一二wrlPDPBO?二 0(】回 INTRODUCTIONmolecular basis of Alzheimer's disease.In Alzheimer/s disease, the p-amyloid peptide Ap assemblesto form a multitude of soluble oligomers as well as insolublefibrils.1,2 The Ap oligomers have emerged as neurotoxicagents that lead to neurodegeneration in Alzheimer's disease.The heterogeneity and metastability of the Ap oligomerspresents a tremendous challenge in understanding themolecular basis of Alzheimer's disease. Specifically, the lackof homogeneous oligomers precludes detailed con elation ofthe biological properties of A0 oligomers with theirstructural and biophysical properties.To reduce the heterogeneily among assemblies of the APpeptide, researchers have prepared and studied Ap oligomersthat consist of A0 monomers linked by chemical cross-links.3"9 These studies have helped determine the importanceof different residues in A(3 oligomerization and havedemonstrated that Ap4()and Ap42 form different types ofoligomers. Crosslinked oligomers have been found to betoxic toward rat pheochromocytoma (PC 12) cells and toinhibit long-term potentiation in rats, providing evidence forthe role of A0 oligomers in neurodegeneration inAlzheimer's disease. Although cross-linking A0 decreasesthe heterogeneity of Ap oligomers, cross-linking has not yetproduced structurally homogeneous oligomers. The high-resolution structures of the cross-linked oligomers that havebeen generated thus fan through either a single disulfide bondor through photoinduced cross-linking of unmodifiedproteins (PICUP), remain unknown.In the past couple of years, our laboratory has identifiedand elucidated hitherto undiscovered modes ofsupramolecularACS Publications© 2016 American Chemical Societyassembly of macrocyclic 0-sheet peptides derived fromamyloidogenic peptides and proteins.111-13 We previouslyreported the X-ray crystallographic structures of twohomologous trimers formed by two macrocyclic p-sheetpeptides derived from A(317-36.10,14 These peptides containAp 17-23 and AB30-36 p-strands covalently linked by two 6-linked ornithine(sOrn) turn mimics and are designed to mimic an A P17-36 (3-15hairpin. Figure 1 illustrates these peptides, 1 and 2, andshows their relationship to an Api7-36 p-hairpin. The 6Ornthat connects residues D23 and A30 replaces the AP24-29 loop;the 6Orn that connects residues L17 and V36 reinforces (3-sheet structure.16 Wc incorporated ornithine (a-linkcd) as ahydrophilic isostere of methionine at position 35 to improvethe solubility of the peptides.14 Peptides 1 and 2 both containan N-methyl group to block uncontrolled aggregation:peptide 1 contains an N-methyl group on G33； peptide 2contains an N methyl group on F20.X-ray crystal