The accumulation of beta-sheet-rich amyloid fibrils or aggregates is a complex, multistep process that is associated with cellular toxicity in a number of human protein misfolding disorders, including Parkinson's and Alzheimer's diseases. It involves the formation of various transient and intransient, on- and off-pathway aggregate species, whose structure, size and cellular toxicity are largely unclear. Here we demonstrate redirection of amyloid fibril formation through the action of a small molecule, resulting in off-pathway, highly stable oligomers. The polyphenol (-)-epigallocatechin gallate efficiently inhibits the fibrillogenesis of both alpha-synuclein and amyloid-beta by directly binding to the natively unfolded polypeptides and preventing their conversion into toxic, on-pathway aggregation intermediates. Instead of beta-sheet-rich amyloid, the formation of unstructured, nontoxic alpha-synuclein and amyloid-beta oligomers of a new type is promoted, suggesting a generic effect on aggregation pathways in neurodegenerative diseases.

Protein misfolding and formation of β-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits α-synuclein and amyloid-β fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature α-synuclein and amyloid-β fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to β-sheetrich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.Alzheimer | Parkinson | catechine | misfolding | oligomer P revious studies have shown that the polyphenol (-)-epi-gallocatechine gallate (EGCG), found in large amounts in green tea, has antiamyloidogenic properties and modulates the misfolding of disease proteins and prions (1-5). EGCG directly binds to unfolded polypeptide chains and inhibits β-sheet formation, an early event in the amyloid formation cascade (6). In the presence of EGCG, the assembly of a new type of unstructured, SDSstable, nontoxic oligomer was observed, instead of the expected formation of β-sheet-rich aggregates. This suggested that the compound redirects aggregation prone polypeptides into offpathway protein assemblies (6), as has since been confirmed for other flavonoids (7).These findings raise the question of whether EGCG might also be able to disassemble preformed, β-sheet-rich structures as well as earlier intermediates of fibrillogenesis. Other small molecules such as curcumin or short β-sheet breaker peptides were described to have this ability; however, their mechanism of action has not been elucidated (8,9). In the present study, we examined the ability of EGCG to alter the structure of mature amyloid fibrils with biochemical and biophysical as well as cell-based assays. Results and DiscussionTo study the effect of EGCG on preformed amyloid aggregates, we first produced α-synuclein (αS) fibrils by incubating natively unfolded monomers (100 μM) at 37°C for 7 d in phosphate buffer. Then aggregates were characterized by EM, atomic force microscopy (AFM), Thioflavin T (ThT) binding assays, and CD spectroscopy (Fig. S1). We observed that the in vitro generated αS aggregates have a β-sheet structure and a fibrillar morphology. Moreover, they efficiently bind the dye ThT, supporting previously published results (10).Next, we added an equimolar concentration of EGCG to the fibrils (50 μM αS monomer equivalent). The effect of the compound was monitored by time-resolved EM and AFM. We found that EGCG very efficiently remodels the ordered, fibrillar morphology ...

Aberrant protein aggregation is a common feature of late-onset neurodegenerative diseases, including Alzheimer's disease, which is associated with the misassembly of the Abeta(1-42) peptide. Aggregation-mediated Abeta(1-42) toxicity was reduced in Caenorhabditis elegans when aging was slowed by decreased insulin/insulin growth factor-1-like signaling (IIS). The downstream transcription factors, heat shock factor 1, and DAF-16 regulate opposing disaggregation and aggregation activities to promote cellular survival in response to constitutive toxic protein aggregation. Because the IIS pathway is central to the regulation of longevity and youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated proteotoxicity.

Several lines of evidence indicate that pre-fibrillar assemblies of amyloid- (A)polypeptides

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and͞or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I ␤-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.␤-turn ͉ ligand binding ͉ protein folding ͉ ␤-sheet ͉ protein function G lobular proteins evolve by mutation and selection. Selection criteria include function, and sufficient thermodynamic stability and folding rate to avoid sustained chaperone binding and proteasome degradation. The selection criteria cannot always be optimized independently over the entire sequence of a protein. For the human Pin1 (hPin1) WW domain (Pin WW hereafter), we have shown that residues important for stability and folding rate are segregated in the sequence (1-4). It is likely that functional selection criteria are predominant once minimal energetic criteria are met. Therefore, sequence evolution to enhance function may lead to a decrease in protein stability and folding rate compared with a sequence optimized for folding energetics.The hPin1 cell cycle regulatory proline (Pro) cis͞trans-isomerase is a two-domain protein (5). In its physiological role, the N-terminal WW domain binds Pro-rich ligands of the consensus sequence (pS͞pT)P, whereas the C-terminal domain catalyzes the Pro cis͞ trans-isomerization at the pS͞pT-P peptide bond. NMR solution studies show that the two domains, which are connected by a flexible solvated linker, interact only weakly before ligand binding (6, 7). The structure of the isolated Pin WW domain is virtually superimposable on that of the WW domain in the two-domain hPin1 protein (8). Moreover, Pin WW exhibits sufficient thermodynamic stability for biophysical analysis, folds rapidly, and retains its ligand-binding function (3, 9). These attributes, combined with sequence information on Ͼ150 WW domain family members (10, 11), makes Pin WW an excellent small model p...

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify A␤, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD. Anfinsen's classic experiments demonstrated that a protein's amino acid sequence specifies its conformation (1). These ideas were extended to explain the misfolding susceptibility of mutant proteins associated with a growing number of familial amyloid diseases (2-5). Although it is thus clear why mutant proteins might be more susceptible to misfolding, it is harder to understand why a wild-type protein or peptide adopts a native conformation in some individuals but it misfolds in others in what should be a common extracellular environment, leading to sporadic amyloid diseases. This discrepancy suggests that other events likely trigger misfolding in sporadic amyloid disease, but their nature remains elusive.The misfolding of secreted amyloid ␤ peptides (A␤) 39-43 residues in length is linked by a plethora of evidence to the pathology of Alzheimer's disease (AD) (6, 7). A␤ misfolding occurs when the soluble, monomeric, extracellular ensemble of extended conformations and low M r oligomers is transformed first into spherical assemblies, then into a number of intermediates, and lastly into fibrillar cross ␤-sheet quaternary structures known as amyloid (8)(9)(10)(11)(12). Amyloid fibrils and related structures recruit soluble A␤ to the aggregate by a seeded polymerization mechanism (10). The direct neurotoxicity of A␤ aggregates (8, 13) combined with their role in mediating chronic inflammation by microglia (14) and complement cascade activation (15) suggests that aggregation then mediates inflammation (16), which in turn promotes aggregation, in a vicious cycle of AD pathology.It is known that atherosclerosis and AD share many risk factors, including hypercholesterolemia and inflammation. The apoE-4 allele, which exacerbates hypercholesterolemia, has been linked to AD by data from both epidemiological and transgenic mouse studies (17)(18)(19)(20). It has also recently been shown t...

Background:It is unknown what the minimum assembly of Tau is that can trigger cell uptake and seeding of intracellular aggregation. Results: Recombinant and AD-derived Tau assemblies were fractionated, and uptake and intracellular seeding activities were determined. Conclusion: Only Tau assemblies of n Ն 3 units trigger uptake and seeding. Significance: Definition of the minimal Tau propagation unit elucidates disease mechanisms for diagnosis and therapy.

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