Increased Aggregation Tendency of Alpha-Synuclein in a Fully Disordered Protein Complex

The recent discovery of biologically active fully disordered, so called random fuzzy protein–protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding.We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn.Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.     

The cellular modifier MOAG‐4/SERF drives amyloid formation through charge complementation

While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.     

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

Prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein (aSyn), a key protein involved in development of Parkinson disease and other synucleinopathies. PREP inhibitors reduce aSyn aggregation, but the mechanism has remained unknown. We have now used protein-fragment complementation assays (PCA) and microscale thermophoresis in parallel to show that PREP interacts directly with aSyn in both intact cells and in a cell-free system. Using split luciferase-based PCA, we first showed that PREP enhances the formation of soluble aSyn dimers in live Neuro-2A neuroblastoma cells. A PREP inhibitor, KYP-2047, reduced aSyn dimerization in PREP-expressing cells but not in cells lacking PREP expression. aSyn dimerization was also enhanced by PREP(S554A), an enzymatically inactive PREP mutant, but this was not affected by KYP-2047. PCA and microscale thermophoresis studies showed that aSyn interacts with both PREP and PREP(S554A) with low micromolar affinity. Neither the proline-rich, C-terminal domain of aSyn nor the hydrolytic activity of PREP was required for the interaction with PREP. Our results show that PREP binds directly to aSyn to enhance its dimerization and may thus serve as a nucleation point for aSyn aggregation. Native gel analysis showed that KYP-2047 shifts PREP to a compact monomeric form with reduced ability to promote aSyn nucleation. As PREP inhibition also enhances autophagic clearance of aSyn, PREP inhibitors may reduce accumulation of aSyn inclusions via a dual mechanism and are thus a novel therapeutic candidate for synucleinopathies. Our results also suggest that PREP has other cellular functions in addition to its peptidase activity.     

Molecular characterization of an aggregation-prone variant of alpha-synuclein used to model synucleinopathies

The misfolding and aggregation of alpha-synuclein (aSyn) are thought to be central events in synucleinopathies. The physiological function of aSyn has been related to vesicle binding and trafficking, but the precise molecular mechanisms leading to aSyn pathogenicity are still obscure. In cell models, aSyn does not readily aggregate, even upon overexpression. Therefore, cellular models that enable the study of aSyn aggregation are essential tools for our understanding of the molecular mechanisms that govern such processes. Here, we investigated the structural features of SynT, an artificial variant of aSyn that has been widely used as a model of aggregation in mammalian cell systems, since it is more prone to aggregation than aSyn. Using Nuclear Magnetic Resonance (NMR) spectroscopy we performed a detailed structural characterization of SynT through a systematic comparison with normal, unmodified aSyn. Interestingly, we found that the conformations adopted by SynT resemble those described for the unmodified protein, demonstrating the usefulness of SynT as a model for aSyn aggregation. However, subtle differences were observed at the N-terminal region involving transient intra and/or intermolecular interactions that are known to regulate aSyn aggregation. Importantly, our results indicate that disturbances in the N-terminal region of SynT, and the consequent decrease in membrane binding of the modified protein, might contribute to the observed aggregation behavior of aSyn, and validate the use of SynT, one of the few models of aSyn aggregation in cultured cells.     

The role of the acidic domain of α-synuclein in amyloid fibril formation: a molecular dynamics study

The detailed mechanism of the pathology of α-synuclein in the Parkinson’s disease has not been clearly elucidated. Recent studies suggested a possible chaperone-like role of the acidic C-terminal region of α-synuclein in the formation of amyloid fibrils. It was also previously demonstrated that the α-synuclein amyloid fibril formation is accelerated by mutations of proline residues to alanine in the acidic region. We performed replica exchange molecular dynamics simulations of the acidic and nonamyloid component (NAC) domains of the wild type and proline-to-alanine mutants of α-synuclein under various conditions. Our results showed that structural changes induced by a change in pH or an introduction of mutations lead to a reduction in mutual contacts between the NAC and acidic regions. Our data suggest that the highly charged acidic region of α-synuclein may act as an intramolecular chaperone by protecting the hydrophobic domain from aggregation. Understanding the function of such chaperone-like parts of fibril-forming proteins may provide novel insights into the mechanism of amyloid formation.     

Assessing the Subcellular Dynamics of Alpha-synuclein Using Photoactivation Microscopy

Alpha-synuclein (aSyn) is implicated in Parkinson’s disease and several other neurodegenerative disorders. To date, the function and intracellular dynamics of aSyn are still unclear. Here, we tracked the dynamics of aSyn using photoactivatable green fluorescent protein as a reporter. We found that the availability of the aSyn N terminus modulates its shuttling into the nucleus. Interestingly, familial aSyn mutations altered the dynamics at which the protein distributes throughout the cell. Both the A30P and A53T aSyn mutations increase the speed at which the protein moves between the nucleus and cytoplasm, respectively. We also found that specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. A mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting phosphorylation modulates aggregation in addition to modulating aSyn intracellular dynamics. Finally, we found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment.     

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer’s disease and Parkinson’s disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD⁺ synthase–nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid β, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.     

shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation,Secretion and Toxicity

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.     

Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer's beta(25-35) amyloid fibril

Amyloid-induced aggregation and precipitation of soluble proteins were investigated in vitro using the amyloid fibrils of the beta(25--35) peptide, a cytotoxic fragment of the Alzheimer's beta-peptide at positions 25--35. The aggregation rate of firefly luciferase was found to be modulated by both a chaperone molecule DnaK and the beta(25--35) amyloid, but their effects were opposite in direction. The amyloid fibril drastically facilitated the luciferase aggregation, which may define a kind of anti-chaperone activity. The effect of the beta(25--35) amyloid to promote protein aggregation and precipitation was further demonstrated for a wide variety of target proteins. The amount of coprecipitation was well correlated with the predicted isoelectric point of the target proteins, indicating that the interaction between the beta(25--35) amyloid and the target was driven by an electrostatic force between them. This view was confirmed by the experiments using an electrically neutral mutant peptide, beta(25--35)KA. It was also found that clustering of the beta(25--35) peptide to form amyloid and the conformation of the target protein are additional factors that determine the strength of the amyloid-protein interaction. Spectroscopic and electron microscopic methods have revealed that the proteins coprecipitated with the beta(25--35) amyloid formed amorphous aggregates deposited together with the amyloid fibrils. The conformation of protein molecules left in the residual soluble fraction was also damaged in the amyloid-containing solution. As a summary, this study has proposed a scheme for events related to the nonspecific amyloid-protein interaction, which may play substantial roles in in vivo conditions.     

Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.     

Amyloid oligomer conformation in a group of natively folded proteins

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic beta-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-beta-aggregation activity in terms of both functionality and in contrast to the beta-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-beta-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-beta-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Abeta aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the beta-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the beta-sheet edge in some natively folded amyloid oligomers is designed positively to prevent beta aggregation.     

FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF*

Patients with Parkinson''s disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.     

The effects of the flavonoid baicalein and osmolytes on the Mg 2+ accelerated aggregation/fibrillation of carboxymethylated bovine 1SS-alpha-lactalbumin

Many protein conformational diseases arise when proteins form alternative stable conformations, resulting in aggregation and accumulation of the protein as fibrillar deposits, or amyloids. Interestingly, numerous proteins implicated in amyloid protein formation show similar structural and functional properties. Given this similarity, we tested the notion that carboxymethylated bovine alpha-lactalbumin (1SS-alpha-lac) could serve as a general amyloid fibrillation/aggregation model system. Like most amyloid forming systems, Mg2+ ions accelerate 1SS-alpha-lac amyloid fibril formation. While osmolytes such as trimethylamine N-oxide (TMAO), and sucrose enhanced thioflavin T detected aggregation, a mixture of trehalose and TMAO substantially inhibited aggregation. Most importantly however, the flavonoid, baicalein, known to inhibit alpha-synuclein amyloid fibril formation, also inhibits 1SS-alpha-lac amyloid with the same apparent efficacy. These data suggest that the easily obtainable 1SS-alpha-lac protein can serve as a general amyloid model and that some small molecule amyloid inhibitors may function successfully with many different amyloid systems.     

Small heat shock proteins protect against alpha-synuclein-induced toxicity and aggregation

Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). Alpha-synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and alphaB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are approximately 2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by approximately 80% in a culture model while alphaB-crystallin reduces toxicity by approximately 20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.     

Kinetically competing huntingtin aggregation pathways control amyloid polymorphism and properties

In polyglutamine (polyQ) containing fragments of the Huntington's disease protein huntingtin (htt), the N-terminal 17 amino acid htt(NT) segment serves as the core of α-helical oligomers whose reversible assembly locally concentrates the polyQ segments, thereby facilitating polyQ amyloid nucleation. A variety of aggregation inhibitors have been described that achieve their effects by neutralizing this concentrating function of the htt(NT) segment. In this paper we characterize the nature and limits of this inhibition for three means of suppressing htt(NT)-mediated aggregation. We show that the previously described action of htt(NT) peptide-based inhibitors is solely due to their ability to suppress the htt(NT)-mediated aggregation pathway. That is, under htt(NT) inhibition, nucleation of polyQ amyloid formation by a previously described alternative nucleation mechanism proceeds unabated and transiently dominates the aggregation process. Removal of the bulk of the htt(NT) segment by proteolysis or mutagenesis also blocks the htt(NT)-mediated pathway, allowing the alternative nucleation pathway to dominate. In contrast, the previously described immunoglobulin-based inhibitor, the antihtt(NT) V(L) 12.3 protein, effectively blocks both amyloid pathways, leading to stable accumulation of nonamyloid oligomers. These data show that the htt(NT)-dependent and -independent pathways of amyloid nucleation in polyQ-containing htt fragments are in direct kinetic competition. The results illustrate how amyloid polymorphism depends on assembly mechanism and kinetics and have implications for how the intracellular environment can influence aggregation pathways.     

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

Implication of novel biochemical property of beta-amyloid

Alzheimer disease (AD) is a heterogeneous disorder with a variety of molecular pathologies converging predominantly on abnormal amyloid deposition particularly in the brain. beta-Amyloid aggregation into senile plaques is one of the pathological hallmarks of AD. beta-Amyloid is generated by a proteolytic cleavage of a large membrane protein, amyloid precursor protein (APP). We have observed a new property of beta-amyloid. The amyloid 1-42 beta fragment, when aggregated, possesses proteolytic and esterase-like activity, in vitro. Three independent methods were used to test the new property of beta-amyloid. While esterase activity involves imidazole catalysis, proteolytic activity is consistent with participation of a serine peptidase triad: catalytic Ser, His and Glu (or Asp). Although the amino acid triad is a necessary requirement for the protease reactivity, it is not sufficient since the secondary structure of the protein significantly contributes to the proteolytic activity. The ability of beta-amyloid to cleave peptide or ester bonds could be thus responsible for either inactivation of other proteins and/or APP proteolysis itself. This property may be responsible for early pathogenesis of AD since there is emerging evidence that non-plaque amyloid is elevated in Alzheimer patients.     

Mechanism of the Chaperone-like and Antichaperone Activities of Amyloid Fibrils of Peptides from αA-Crystallin

The amyloid fibril of a fragment of the substrate binding site of αA-crystallin (αAC(71-88)) exhibited chaperone-like activity by suppressing the aggregation of alcohol dehydrogenase (ADH) and luciferase. By contrast, the amyloid fibril of the cytotoxic fragment of amyloid β protein (Aβ(25-35)) facilitated the aggregation of the same proteins. We have determined the zeta potential of the amyloid fibril by measuring their electrophoretic mobility to study the effects of the surface charge on the modulation of protein aggregation. The αAC(71-88) amyloid possesses a large negative zeta potential value which is unaffected by the binding of the negatively charged ADH, indicating that the αAC(71-88) amyloid is stable as a colloidal dispersion. By contrast, the Aβ(25-35) amyloid possesses a low zeta potential value, which was significantly reduced with the binding of the negatively charged ADH. The canceling of the surface charge of the amyloid fibril upon substrate binding reduces colloidal stability and thereby facilitates protein aggregation. These results indicate that one of the key factors determining whether amyloid fibrils display chaperone-like or antichaperone activity is their electrostatic interaction with the substrate. The surface of the αAC(71-88) amyloid comprises a hydrophobic environment, and the chaperone-like activity of the αAC(71-88) amyloid is best explained by the reversible substrate binding driven by hydrophobic interactions. On the basis of these findings, we designed variants of amyloid fibrils of αAC(71-88) that prevent protein aggregation associated with neurodegenerative disorders.     

The effect of small molecules in modulating the chaperone activity of alphaB-crystallin against ordered and disordered protein aggregation

Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as alphaB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of alphaB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of alphaB-crystallin against aggregation of the disease-related amyloid fibril forming protein alpha-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.