Effects of the Polyphenols Delphinidin and Rosmarinic Acid on the Inducible Intra-cellular Aggregation of Alpha-Synuclein in Model Neuron Cells.

Intracellular aggregation of α-synuclein is a major pathological feature of Parkinson's disease. In this study, we show that the polyphenols delphinidin and rosmarinic acid suppress intracellular aggregation of α-synuclein in a mouse neuron cell model when added under oxidative stress conditions. To enhance the detection threshold of this preventive effect of the two polyphenols, we generated a new strain of "aggregation prone model cells" that tended to show prominent α-synuclein aggregation even under normal conditions. Using this new highly sensitive cell line, we demonstrate that addition of delphinidin to model cell cultures effectively suppresses the formation of intracellular α-synuclein aggregates. Flow cytometric analysis shows that adding delphinidin decreases the fraction of "dying cells," cells that were alive but in a damaged state. Our findings suggest the possibility of using polyphenols to prevent and treat the symptoms correlated with the onset of Parkinson's disease. Additionally, our aggregation-prone cell model may be used in future studies to probe numerous neurodegenerative diseases with high sensitivity.

An α-synuclein decoy peptide prevents cytotoxic α-synuclein aggregation caused by fatty acid binding protein 3.

α-synuclein (αSyn) is a protein known to form intracellular aggregates during the manifestation of Parkinson's disease. Previously, it was shown that αSyn aggregation was strongly suppressed in the midbrain region of mice that did not possess the gene encoding the lipid transport protein fatty acid binding protein 3 (FABP3). An interaction between these two proteins was detected in vitro, suggesting that FABP3 may play a role in the aggregation and deposition of αSyn in neurons. To characterize the molecular mechanisms that underlie the interactions between FABP3 and αSyn that modulate the cellular accumulation of the latter, in this report, we used in vitro fluorescence assays combined with fluorescence microscopy, transmission electron microscopy, and quartz crystal microbalance assays to characterize in detail the process and consequences of FABP3-αSyn interaction. We demonstrated that binding of FABP3 to αSyn results in changes in the aggregation mechanism of the latter; specifically, a suppression of fibrillar forms of αSyn and also the production of aggregates with an enhanced cytotoxicity toward mice neuro2A cells. Because this interaction involved the C-terminal sequence region of αSyn, we tested a peptide derived from this region of αSyn (αSynP130-140) as a decoy to prevent the FABP3-αSyn interaction. We observed that the peptide competitively inhibited binding of αSyn to FABP3 in vitro and in cultured cells. We propose that administration of αSynP130-140 might be used to prevent the accumulation of toxic FABP3-αSyn oligomers in cells, thereby preventing the progression of Parkinson's disease.

Spearmint Extract Containing Rosmarinic Acid Suppresses Amyloid Fibril Formation of Proteins Associated with Dementia.

Neurological dementias such as Alzheimer's disease and Lewy body dementia are thought to be caused in part by the formation and deposition of characteristic insoluble fibrils of polypeptides such as amyloid beta (Aß), Tau, and/or α-synuclein (αSyn). In this context, it is critical to suppress and remove such aggregates in order to prevent and/or delay the progression of dementia in these ailments. In this report, we investigated the effects of spearmint extract (SME) and rosmarinic acid (RA; the major component of SME) on the amyloid fibril formation reactions of αSyn, Aß, and Tau proteins in vitro. SME or RA was added to soluble samples of each protein and the formation of fibrils was monitored by thioflavin T (ThioT) binding assays and transmission electron microscopy (TEM). We also evaluated whether preformed amyloid fibrils could be dissolved by the addition of RA. Our results reveal for the first time that SME and RA both suppress amyloid fibril formation, and that RA could disassemble preformed fibrils of αSyn, Aß, and Tau into non-toxic species. Our results suggest that SME and RA may potentially suppress amyloid fibrils implicated in the progression of Alzheimer's disease and Lewy body dementia in vivo, as well.

Acid-denatured small heat shock protein HdeA from Escherichia coli forms reversible fibrils with an atypical secondary structure.

The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a "reversible" form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its "molecular chaperone" activity) and the formation of inactive fibrillary deposits (its "fibrillogenic" activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail.

Common structural features of toxic intermediates from α-synuclein and GroES fibrillogenesis detected using cryogenic coherent X-ray diffraction imaging.

The aggregation and deposition of α-synuclein (αSyn) in neuronal cells is correlated to pathogenesis of Parkinson's disease. Although the mechanism of αSyn aggregation and fibril formation has been studied extensively, the structural hallmarks that are directly responsible for toxicity toward cells are still under debate. Here, we have compared the structural characteristics of the toxic intermediate molecular species of αSyn and similar toxic species of another protein, GroES, using coherent X-ray diffraction analysis. Using coherent X-ray free electron laser pulses of SACLA, we analysed αSyn and GroES fibril intermediate species and characterized various aggregate structures. Unlike previous studies where an annular oligomeric form of αSyn was identified, particle reconstruction from scattering traces suggested that the specific forms of the toxic particles were varied, with the sizes of the particles falling within a specific range. We did however discover a common structural feature in both αSyn and GroES samples; the edges of the detected particles were nearly parallel and produced a characteristic diffraction pattern in the diffraction experiments. The presence of parallel-edged particles in toxic intermediates of αSyn and GroES fibrillogenesis pointed towards a plausible common molecular interface that leads to the formation of mature fibrils.

Modulating the Effects of the Bacterial Chaperonin GroEL on Fibrillogenic Polypeptides through Modification of Domain Hinge Architecture.

The isolated apical domain of the Escherichia coli GroEL subunit displays the ability to suppress the irreversible fibrillation of numerous amyloid-forming polypeptides. In previous experiments, we have shown that mutating Gly-192 (located at hinge II that connects the apical domain and the intermediate domain) to a tryptophan results in an inactive chaperonin whose apical domain is disoriented. In this study, we have utilized this disruptive effect of Gly-192 mutation to our advantage, by substituting this residue with amino acid residues of varying van der Waals volumes with the intent to modulate the affinity of GroEL toward fibrillogenic peptides. The affinities of GroEL toward fibrillogenic polypeptides such as Aß(1-40) (amyloid-ß(1-40)) peptide and α-synuclein increased in accordance to the larger van der Waals volume of the substituent amino acid side chain in the G192X mutants. When we compared the effects of wild-type GroEL and selected GroEL G192X mutants on α-synuclein fibril formation, we found that the effects of the chaperonin on α-synuclein fibrillation were different; the wild-type chaperonin caused changes in both the initial lag phase and the rate of fibril extension, whereas the effects of the G192X mutants were more specific toward the nucleus-forming lag phase. The chaperonins also displayed differential effects on α-synuclein fibril morphology, suggesting that through mutation of Gly-192, we may induce changes to the intermolecular affinities between GroEL and α-synuclein, leading to more efficient fibril suppression, and in specific cases, modulation of fibril morphology.

Structural basis of Cu, Zn-superoxide dismutase amyloid fibril formation involves interaction of multiple peptide core regions.

Cu, Zn-superoxide dismutase (SOD1), an enzyme implicated in the progression of familial amyotrophic lateral sclerosis (fALS), forms amyloid fibrils under certain experimental conditions. As part of our efforts to understand ALS pathogenesis, in this study we found that reduction of the intramolecular disulfide bond destabilized the tertiary structure of metal free wild-type SOD1 and greatly enhanced fibril formation in vitro. We also identified fibril core peptides that are resistant to protease digestion by using mass spectroscopy and Edman degradation analyses. Three regions dispersed throughout the sequence were detected as fibril core sequences of SOD1. Interestingly, by using three synthetic peptides that correspond to these identified regions, we determined that each region was capable of fibril formation, either alone or in a mixture containing multiple peptides. It was also revealed that by reducing the disulfide bond and causing a decrease in the structural stability, the amyloid fibril formation of a familial mutant SOD1 G93A was accelerated even under physiological conditions. These results demonstrate that by destabilizing the structure of SOD1 by removing metal ions and breaking the intramolecular disulfide bridge, multiple fibril-forming core regions are exposed, which then interact with each another and form amyloid fibrils under physiological conditions.

Anthocyanin suppresses the toxicity of Aß deposits through diversion of molecular forms in in vitro and in vivo models of Alzheimer's disease.

OBJECTIVES: The pathogenesis of Alzheimer's disease (AD) is strongly correlated with the aggregation and deposition of the amyloid beta (Aß1-42) peptide in fibrillar form, and many studies have shown that plant-derived polyphenols are capable of attenuating AD progression in various disease models. In this study, we set out to correlate the effects of anthocyanoside extracts (Vaccinium myrtillus anthocyanoside (VMA)) obtained from bilberry on the in vitro progression of Aß fibril formation with the in vivo effects of this compound on AD pathogenesis. METHODS: Thioflavin T fluorescence assays and atomic force microscopy were used to monitor Aß amyloid formation in in vitro assays. Effects of Aß amyloids on cellular viability were assayed using cultured Neuro2a cells. Cognitive effects were probed using mice that simultaneously expressed mutant human Aß precursor and mutant presenilin-2. RESULTS: Addition of VMA inhibited the in vitro formation of Aß peptide fibrils and also reduced the toxicity of these aggregates toward Neuro2a cells. A diet containing 1% VMA prevented the cognitive degeneration in AD mice. Curiously, this diet-derived retention of cognitive ability was not accompanied by a reduction in aggregate deposition in brains; rather, an increase in insoluble deposits was observed compared with mice raised on a control diet. DISCUSSION: The paradoxical increase in insoluble deposits caused by VMA suggests that these polyphenols divert Aß aggregation to an alternate, non-toxic form. This finding underscores the complex effects that polyphenol compounds may exert on amyloid deposition in vivo.

Bilberry anthocyanins neutralize the cytotoxicity of co-chaperonin GroES fibrillation intermediates.

The co-chaperonin GroES (Hsp10) works with chaperonin GroEL (Hsp60) to facilitate the folding reactions of various substrate proteins. Upon forming a specific disordered state in guanidine hydrochloride, GroES is able to self-assemble into amyloid fibrils similar to those observed in various neurodegenerative diseases. GroES therefore is a suitable model system to understand the mechanism of amyloid fibril formation. Here, we determined the cytotoxicity of intermediate GroES species formed during fibrillation. We found that neuronal cell death was provoked by soluble intermediate aggregates of GroES, rather than mature fibrils. The data suggest that amyloid fibril formation and its associated toxicity toward cell might be an inherent property of proteins irrespective of their correlation with specific diseases. Furthermore, with the presence of anthocyanins that are abundant in bilberry, we could inhibit both fibril formation and the toxicity of intermediates. Addition of bilberry anthocyanins dissolved the toxic intermediates and fibrils, and the toxicity of the intermediates was thus neutralized. Our results suggest that anthocyanins may display a general and potent inhibitory effect on the amyloid fibril formation of various conformational disease-causing proteins.

Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins.

Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.

Probing the functional mechanism of Escherichia coli GroEL using circular permutation.

BACKGROUND: The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit. METHODOLOGY/PRINCIPAL FINDINGS: Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion. CONCLUSIONS/SIGNIFICANCE: The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.

Covalent structural changes in unfolded GroES that lead to amyloid fibril formation detected by NMR: insight into intrinsically disordered proteins.

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn(45)-Gly(46) and/or Asn(51)-Gly(52) that eventually yield ß-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.

A potentially versatile nucleotide hydrolysis activity of group II chaperonin monomers from Thermoplasma acidophilum.

Compared to the group I chaperonins such as Escherichia coli GroEL, which facilitate protein folding, many aspects of the functional mechanism of archaeal group II chaperonins are still unclear. Here, we show that monomeric forms of archaeal group II chaperonin alpha and beta from Thermoplasma acidophilum may be purified stably and that these monomers display a strong AMPase activity in the presence of divalent ions, especially Co(2+) ion, in addition to ATPase and ADPase activities. Furthermore, other nucleoside phosphates (guanosine, cytidine, uridine, and inosine phosphates) in addition to adenine nucleotides were hydrolyzed. From analyses of the products of hydrolysis using HPLC, it was revealed that the monomeric chaperonin successively hydrolyzed the phosphoanhydride and phosphoester bonds of ATP in the order of gamma to alpha. This activity was strongly suppressed by point mutation of specific essential aspartic acid residues. Although these archaeal monomeric chaperonins did not alter the refolding of MDH, their novel versatile nucleotide hydrolysis activity might fulfill a new function. Western blot experiments demonstrated that the monomeric chaperonin subunits were also present in lysed cell extracts of T. acidophilum, and partially purified native monomer displayed Co(2+)-dependent AMPase activity.

Gly192 at hinge 2 site in the chaperonin GroEL plays a pivotal role in the dynamic apical domain movement that leads to GroES binding and efficient encapsulation of substrate proteins.

The subunit structure of chaperonin GroEL is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The 'hinge 2' site of GroEL contains three glycine residues, Gly192, Gly374, and Gly375, connecting the apical domain and the intermediate domain. In this study, to understand the importance of the hinge 2 amino acid residues in chaperonin function, we substituted each of these three glycine residues to tryptophan. The GroEL mutants G374W and G375W were functionally similar to wild-type GroEL. However, GroEL G192W showed a significant decrease in the ability to assist the refolding of stringent substrate proteins. Interestingly, from biochemical assays and characterization using surface plasmon resonance analysis, we found that GroEL G192W was capable of binding GroES even in the absence of ATP to form a very stable GroEL-GroES complex, which could not be dissociated even upon addition of ATP. Electron micrographs showed that GroEL G192W intrinsically formed an asymmetric double ring structure with one ring locked in the 'open' conformation, and it is postulated that GroES binds to this open ring in the absence of ATP. Trans-binding of both substrate protein and GroES was observed for this binary complex, but simultaneous binding of both substrate and GroES (a mechanism that ensures substrate encapsulation) was impaired. We postulate that alteration of Gly192 severely compromises an essential movement that allows efficient encapsulation of unfolded protein intermediates.

Hydrophilic residues 526 KNDAAD 531 in the flexible C-terminal region of the chaperonin GroEL are critical for substrate protein folding within the central cavity.

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.

Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure.

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.

Multiple structural transitions of the GroEL subunit are sensitive to intermolecular interactions with cochaperonin and refolding polypeptide.

In this study we attempted to determine the specific roles of the numerous conformational changes that are observed in the bacterial chaperonin GroEL, by performing stopped-flow experiments on GroEL R231W in the presence of a refolding substrate protein. The apparent rate of one kinetic phase was decreased by approximately 25% in the presence of prebound unfolded malate dehydrogenase while another phase was suppressed completely under the same conditions, reflecting different effects of the unfolded protein on multiple structural transitions within GroEL. The addition of cochaperonin GroES counteracts the effect of the bound substrate protein in the former case, but had no effect on the latter, more extensive suppression. Using a chemically modified form of GroEL R231W which is incapable of releasing substrate proteins at low temperatures, we identified a conformational transition that is implicated in the release of substrate proteins. Parts of the actual process of substrate protein release were also observed through fluorescence resonance energy transfer experiments involving GroEL and labeled substrate protein. Analysis of the energy transfer data revealed an interesting relationship between substrate protein displacement and a specific structural transition in the GroEL apical domain.

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion.

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases.

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.