Cleavage agents for soluble oligomers of human islet amyloid polypeptide

Soluble oligomers of human islet amyloid polypeptide (h-IAPP) are implicated in the initiation of beta-cell apoptosis leading to type 2 diabetes mellitus (T2DM). Cleavage of the h-IAPP included in an oligomer may provide a novel method for reducing the level of h-IAPP oligomers, offering a new therapeutic option for T2DM. From the combinatorial library of triazine derivatives prepared by exploiting the Co(III) complex of cyclen as the cleavage center for peptide bonds, eight compounds were selected as cleavage agents for oligomers of h-IAPP. After reaction with cleavage agents for 36 h at 37 degrees C and pH 7.50, up to 20 mol% of h-IAPP (initial concentration: 4.0 microM) was cleaved, although the target oligomers existed as transient species. Considerable activity was manifested at agent concentrations as low as 100 nM.     

Peptide-cleaving agents for human islet amyloid polypeptide containing substrate recognition site based on quinoxaline: cleavage efficiency enhanced by lowering substrate concentration

Oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type 2 diabetes mellitus. Peptide-cleaving agents selective for oligomers of h-IAPP were synthesized by using quinoxaline derivatives as recognition sites attached to the Co(III) complex of cyclen in this study. When the initial concentration of h-IAPP was lowered from 4.0 to 0.20 μM, cleavage yield of the new agents was enhanced by 3 times reaching 16-22 mol%. This shows that the agents would have significant activities at subnano molar concentrations if the concentration of h-IAPP is lowered to the in vivo values. This further indicates that the peptide-cleaving agents prepared previously in this laboratory possess sufficiently high activity for application as a new therapeutic option for Alzheimer's disease, type 2 diabetes mellitus, and Parkinson's disease.     

New chelating ligands for Co(III)-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses

The Co(III) complex of 1,4,7,10-tetraazacyclododecane has been employed as the catalytic center of target-selective peptide-cleaving catalysts in previous studies. As new chelating ligands for the Co(III) ion in the peptide-cleaving catalysts, 1-oxo-4,7,10-triazacyclodedecane, 1-aryl-1,4,7,10-tetraazacyclodecane, and 7-aryl-1-oxo-4,7,10-triazacyclodecane were examined in the present study. A chemical library comprising 612 derivatives of the Co(III) complex of the new chelating ligands was constructed. The catalyst candidates were tested for their activity to cleave the soluble oligomers of amyloidogenic peptides amyloid β-42 and human islet amyloid polypeptide (h-IAPP), which are believed to be the pathogenic species for Alzheimer’s disease and type 2 diabetes mellitus, respectively. One derivative of the Co(III) complex of 1-aryl-1,4,7,10-tetraazacyclodecane was found to cleave the oligomers of h-IAPP. Cleavage products were identified and cleavage yields were measured at various catalyst concentrations for the action of the new catalyst. The present results reveal that effective catalytic drugs for amyloidoses may be obtained by using Co(III) complexes of various chelating ligands.     

Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

Cu(II) cyclen cleavage agent for human islet amyloid peptide

Type 2 diabetes mellitus (T2DM) is characterized by a substantial reduction in β-cell mass and the amyloid fibrils which are formed by the aggregation of the human islet amyloid polypeptide (h-IAPP) in the islet of Langerhans. Cleavage agents with Co(III) cyclen as the catalytic group have been studied as a novel therapeutic option for T2DM patients. However, recent research has suggested that the cytotoxicity of h-IAPP might be mediated by interactions with Cu(II); furthermore, it has been shown in vitro that Cu(II) prevents h-IAPP from forming the β-sheet conformers. Therefore, we synthesized a cleavage agent using Cu(II) cyclen. The resulting cleaved fragments and estimated cleavage yield (8.3 mol %) were evaluated after incubation with h-IAPP.     

The molecular basis of distinct aggregation pathways of islet amyloid polypeptide

Abnormal aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils is a hallmark of type 2 diabetes. In this study, we investigated the initial oligomerization and subsequent addition of monomers to growing aggregates of human IAPP at the residue-specific level using NMR, atomic force microscopy, mass spectroscopy, and computational simulations. We found that in solution IAPPs rapidly associate into transient low-order oligomers such as dimers and trimers via interactions between histidine 18 and tyrosine 37. This initial event is proceeded by slow aggregation into higher-order spherical oligomers and elongated fibrils. In these two morphologically distinct types of aggregates IAPPs adopt structures with markedly different residual flexibility. Here we show that the anti-amyloidogenic compound resveratrol inhibits oligomerization and amyloid formation via binding to histidine 18, supporting the finding that this residue is crucial for on-pathway oligomer formation.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

The Mechanism of Membrane Disruption by Cytotoxic Amyloid Oligomers Formed by Prion Protein(106–126) Is Dependent on Bilayer Composition

The formation of fibrillar aggregates has long been associated with neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although fibrils are still considered important to the pathology of these disorders, it is now widely understood that smaller amyloid oligomers are the toxic entities along the misfolding pathway. One characteristic shared by the majority of amyloid oligomers is the ability to disrupt membranes, a commonality proposed to be responsible for their toxicity, although the mechanisms linking this to cell death are poorly understood. Here, we describe the physical basis for the cytotoxicity of oligomers formed by the prion protein (PrP)-derived amyloid peptide PrP(106–126). We show that oligomers of this peptide kill several mammalian cells lines, as well as mouse cerebellar organotypic cultures, and we also show that they exhibit antimicrobial activity. Physical perturbation of model membranes mimicking bacterial or mammalian cells was investigated using atomic force microscopy, polarized total internal reflection fluorescence microscopy, and NMR spectroscopy. Disruption of anionic membranes proceeds through a carpet or detergent model as proposed for other antimicrobial peptides. By contrast, when added to zwitterionic membranes containing cholesterol-rich ordered domains, PrP(106–126) oligomers induce a loss of domain separation and decreased membrane disorder. Loss of raft-like domains may lead to activation of apoptotic pathways, resulting in cell death. This work sheds new light on the physical mechanisms of amyloid cytotoxicity and is the first to clearly show membrane type-specific modes of action for a cytotoxic peptide.     

Sulfated glycosaminoglycans accelerate transthyretin amyloidogenesis by quaternary structural conversion

Glycosaminoglycans (GAGs), which are found in association with all extracellular amyloid deposits in humans, are known to accelerate the aggregation of various amyloidogenic proteins in vitro. However, the precise molecular mechanism(s) by which GAGs accelerate amyloidogenesis remains elusive. Herein, we show that sulfated GAGs, especially heparin, accelerate transthyretin (TTR) amyloidogenesis by quaternary structural conversion. The clustering of sulfate groups on heparin and its polymeric nature are essential features for accelerating TTR amyloidogenesis. Heparin does not influence TTR tetramer stability or TTR dissociation kinetics, nor does it alter the folded monomer-misfolded monomer equilibrium directly. Instead, heparin accelerates the conversion of preformed TTR oligomers into larger aggregates. The more rapid disappearance of monomeric TTR in the presence of heparin likely reflects the fact that the monomer-misfolded amyloidogenic monomer-oligomer-TTR fibril equilibria are all linked, a hypothesis that is strongly supported by the light scattering data. TTR aggregates prepared in the presence of heparin exhibit a higher resistance to trypsin and proteinase K proteolysis and a lower exposure of hydrophobic side chains comprising hydrophobic clusters, suggesting an active role for heparin in amyloidogenesis. Our data suggest that heparin accelerates TTR aggregation by a scaffold-based mechanism, in which the sulfate groups comprising GAGs interact primarily with TTR oligomers through electrostatic interactions, concentrating and orienting the oligomers, facilitating the formation of higher molecular weight aggregates. This model raises the possibility that GAGs may play a protective role in human amyloid diseases by interacting with proteotoxic oligomers and promoting their association into less toxic amyloid fibrils.     

A model of amyloid's role in disease based on fibril fracture

Although the correlative evidence relating the presence of amyloid fibrils and certain disease states (e.g. Alzheimer's disease and Type 2 Diabetes) is overwhelming, a direct causative role for amyloid has proved harder to establish. Current thinking links a narrow region of the aggregate protein size distribution, the so called ‘early aggregate’ domain to cellular toxicity. A troubling feature of this theory however is that the nucleated reaction mechanism by which amyloid formation is believed to occur results in a very low number concentration of early aggregates which are rapidly extended to form amyloid fibrils. This situation means that the concentration of early aggregates is very low at the time when they are supposedly at their most toxic. Here we adopt a novel explicit simulation strategy to examine a kinetic regime involving nucleated growth combined with fibril fragmentation under which this situation can be reversed so as to produce a high number concentration of small on-pathway toxic aggregates. Dependent upon the rate of fragmentation, the time scale for generation of toxic early aggregates may be coupled, uncoupled or disassociated from the time scale for the appearance of amyloid fibrils. Furthermore the model presents itself as a biochemical ‘switch’ transitioning between modes of amyloid induced cell death dependent upon either specific amyloid toxicity or non-specific solid mass induced tissue damage.     

Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Conformation-dependent scFv antibodies specifically recognize the oligomers assembled from various amyloids and show colocalization of amyloid fibrils with oligomers in patients with amyloidoses

Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a na?ve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aβ1-40, prion peptide 106-126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aβ1-40 and prion peptide 106-126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses.     

Human IAPP amyloidogenic properties and pancreatic β-cell death

A hallmark of type 2 diabetes mellitus (T2DM) is the presence of extracellular amyloid deposits in the islets of Langerhans. These deposits are formed by the human islet amyloid polypeptide, hIAPP (or amylin), which is a hormone costored and cosecreted with insulin. Under normal conditions, the hormone remains in solution but, in the pancreas of T2DM individuals, it undergoes misfolding giving rise to oligomers and cross-β amyloid fibrils. Accumulating evidence suggests that the amyloid deposits that accompany type 2 diabetes mellitus are not just a trivial epiphenomenon derived from the disease progression. Rather, hIAPP aggregation induces processes that impair the functionality and viability of β-cells and may lead to apoptosis. The present review article aims to summarize a few aspects of the current knowledge of this amyloidogenic polypeptide. In the first place, the physicochemical properties which condition its propensity to misfold and form aggregates. Secondly, how these properties confer hIAPP the capacity to interfere with some signaling of the pancreatic β-cell, interact with membranes, form channels or affect natural ion channels, including calcium channels. Finally, how misfolded hIAPP cytotoxicity results in apoptosis. A number of pathophysiological changes of the T2DM islet can be related to the amyloidogenic properties of hIAPP. However, in a certain way, the in vivo aggregation of the polypeptide also reflects a failure of chaperones and, in general, of cellular proteostasis, supporting the view that T2DM may also be considered as a conformational disorder.     

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.     

Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways

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 Aβ42 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 Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ 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 Aβ soluble oligomers and related aggregated conformers.     

Role of conformational dynamics in pathogenic protein aggregation

The accumulation of pathogenic protein oligomers and aggregates is associated with several devastating amyloid diseases. As protein aggregation is a multi-step nucleation-dependent process beginning with unfolding or misfolding of the native state, it is important to understand how innate protein dynamics influence aggregation propensity. Kinetic intermediates composed of heterogeneous ensembles of oligomers are frequently formed on the aggregation pathway. Characterization of the structure and dynamics of these intermediates is critical to the understanding of amyloid diseases since oligomers appear to be the main cytotoxic agents. In this review, we highlight recent biophysical studies of the roles of protein dynamics in driving pathogenic protein aggregation, yielding new mechanistic insights that can be leveraged for design of aggregation inhibitors.     

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils: IMPLICATIONS FOR CANCER

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.     

Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein

The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the β-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations.     

Modulation effect of filamentous phage on α-synuclein aggregation

Conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases, including Parkinson’s disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of α-synuclein (AS) in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.     

Conversion of Aβ42 into a Folded Soluble Native-like Protein using a Semi-random Library of Amphipathic Helices

The amyloid cascade model hypothesizes that neurotoxic oligomers or aggregates formed by the Alzheimer amyloid peptide (Aβ) cause disease pathology in Alzheimer's disease. Attempted treatment strategies for Alzheimer's disease have involved either inhibiting Aβ oligomerization or aggregation, or dissolving existing aggregates. Blocking such downhill processes, however, has proved daunting. We have used a different approach that targets Aβ before the oligomerization cascade begins. We predicted that an amphipathic helix could convert Aβ into a native-like protein and inhibit initiation of oligomerization and aggregation. This idea was tested with a designed library and genetic screen. We exhaustively screened a library of semi-randomized amphipathic helical sequences, each expressed as a fusion protein with an Aβ42-yellow fluorescent protein sequence serving as a reporter for folding and solubilization. This yielded an amphipathic helix capable of initiating native-like folding in Aβ42 and preventing aggregation. This amphipathic helix has direct application to Alzheimer's disease therapy development.