Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the A␤1-42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate A␤ monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their ␤-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for A␤-mediated toxicity.Despite the remarkable fidelity of protein folding in diverse cellular environments, defects do occur that are linked to an array of protein aggregation diseases. In many such disorders (e.g. Alzheimer (1-4), Parkinson (5, 6), Huntington (7-9), and Prion (10, 11) diseases) specific peptides of unrelated sequence aggregate into similar types of assemblies ranging from soluble, low molecular weight oligomers to insoluble, high molecular weight amyloid fibrils (1, 12).A particularly intriguing aspect of protein misfolding is that a single polypeptide chain can adopt multiple aggregated conformations with unique biological activities (13). Such conformational diversity was first observed for the mammalian prion protein PrP (14 -21). Different infectious prion conformations of PrP, known as strains or variants, encipher unique prion diseases through differences in their aggregate structure (14,16,19,(22)(23)(24). More recently, polymorphic aggregate structures have been formed in vitro and identified in vivo for many other proteins (25-39). However, the biological consequence of such conformational diversity and which conformers are most toxic remains poorly defined.Aggregated A␤ conformers associated with Alzheimer disease also display such conformational diversity (30,32,33,38,40). The A␤ peptide self-assembles through multiple pathways in which several intermediates are transiently populated (41-46). These conformers, which range from dimers and soluble oligomers to fibrillar oligomers and protofibrils, are typically classified either by size or structure. Even though size is an important characteristic of different A␤ conformers, it is now clear that aggregates of the same size can have unique structures (44, 47). These recent findings have been illumi...

Background:The Alzheimer A␤ peptide assembles into multiple small oligomers that are cytotoxic. Results: Increased solvent exposure of hydrophobic residues within non-fibrillar A␤ oligomers of similar size increases cytotoxicity. Conclusion: A␤ non-fibrillar oligomers display size-independent differences in toxicity that are strongly influenced by oligomer conformation. Significance: Identifying the conformational determinants of A␤-mediated toxicity is critical to understand and treat Alzheimer disease.

Tissue-specific overexpression of the human systemic amyloid precursor transthyretin (TTR) ameliorates Alzheimer's disease (AD) phenotypes in APP23 mice. TTR-␤-amyloid (A␤) complexes have been isolated from APP23 and some human AD brains. We now show that substoichiometric concentrations of TTR tetramers suppress A␤ aggregation in vitro via an interaction between the thyroxine binding pocket of the TTR tetramer and A␤ residues 18 -21 (nuclear magnetic resonance and epitope mapping). The K D is micromolar, and the stoichiometry is Ͻ1 for the interaction (isothermal titration calorimetry). Similar experiments show that engineered monomeric TTR, the best inhibitor of A␤ fibril formation in vitro, did not bind A␤ monomers in liquid phase, suggesting that inhibition of fibrillogenesis is mediated by TTR tetramer binding to A␤ monomer and both tetramer and monomer binding of A␤ oligomers. The thousandfold greater concentration of tetramer relative to monomer in vivo makes it the likely suppressor of A␤ aggregation and disease in the APP23 mice.

In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the A␤42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated A␤. In contrast, a Class II molecule converts soluble A␤ oligomers into fibrils, but is inactive against disaggregated and fibrillar A␤. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, A␤ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than A␤ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel A␤ soluble oligomers and related aggregated conformers.A central tenet of protein folding is that a given amino acid sequence encodes a single folded structure (1). By analogy, one would expect that a given protein sequence would encode a single misfolded structure (e.g. a single amyloid fibril conformation). Instead, each protein sequence encodes numerous aggregated isoforms that possess unique secondary and tertiary structures (2-12). Previous work has firmly established that small, prefibrillar conformers (herein referred to as soluble oligomers) of diverse polypeptides are the most toxic aggregates both in vitro and in vivo (11,(13)(14)(15)(16)(17). However, elucidating the structural attributes of such toxic conformers that differentiate them from their non-toxic counterparts has proven difficult (see Refs. 11 and 18 -22 for recent progress).Significant evidence linking protein misfolding to cellular toxicity in numerous aggregation disorders has motivated the search for small molecules that prevent aggregation (see Refs. 23-25, and references therein). A general conclusion of these studies is that many small molecules redirect the aggregation cascade rather than inhibiting it completely (26). In hind...

Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation-and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6-10 residues) from the Aβ42 peptide associated with Alzheimer's disease into the complementarity determining regions of a domain (V H ) antibody generates antibody variants that recognize Aβ soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aβ motif ( 18 VFFA 21 ) are reactive with Aβ fibrils, whereas those displaying the amyloidogenic C terminus ( 34 LMVGGVVIA 42 ) are reactive with Aβ fibrils and oligomers (and weakly reactive with Aβ monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aβ conformers. Aβ gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aβ soluble oligomers and fibrils also neutralize the toxicity of each Aβ conformer. We expect that our antibody design strategy is not limited to Aβ and can be used to readily generate gammabodies against other toxic misfolded proteins.misfolding | beta-amyloid | protein engineering A hallmark of protein misfolding disorders is that polypeptides of unrelated sequence fold into similar oligomeric and fibrillar assemblies that are cytotoxic (1). The structures of these enigmatic conformers have captured the imagination of many investigators who have sought to explain the molecular basis of proteotoxicity in conformational disorders such as Alzheimer's disease. Because misfolded proteins are typically refractory to structural methods such as X-ray crystallography and solution NMR, few high-resolution structures of full-length misfolded proteins have been reported (ref. 2 and references therein). The structures of oligomeric intermediates have proven especially difficult to characterize because these conformers are labile, transient, and, in many cases, heterogeneous.Given the complexity of high-resolution structural analysis of misfolded proteins, alternative biochemical approaches are critical for understanding structure-function relationships of aggregated proteins. A breakthrough in this area has been the development of conformation-specific antibodies that selectively recognize uniquely folded conformers of amyloidogenic proteins (3-13). Indeed, multiple conformation-specific antibodies have been reported that recognize structural features ...