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'In dopaminergic neurons, alpha-synuclein (alphaS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of alphaS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which alphaS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of alphaS. Exchange between the free state and the lipid-bound alphaS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s(-1). Partitioning of the binding modes is dependent on lipid/alphaS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of alphaS adopts an alpha-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound alphaS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large alpha-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this "dark state." Titration of alphaS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where alphaS lies in an alpha-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by alphaS.

To better model the characteristics of crowded intracellular environments, we examined the effect of several factors known to induce partial folding and accelerated fibrillation of alpha-synuclein in dilute solutions, on the fibrillation of this protein in a crowded milieu. We found that low pH, certain metals and pesticides, polyanions, polycations and low concentrations of organic solvents cause a significant acceleration of alpha-synuclein fibrillation in the presence of high concentrations of polyethylene glycol. This suggests that the fibril-promoting effects of factors inducing partial folding of alpha-synuclein and the fibril-stimulating effects of macromolecular crowding are relatively independent and thus might act additively or even synergistically.

The deposition of alpha-synuclein (alpha-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate alpha-syn aggregation, it is unclear which other factors can regulate the actions of DA on alpha-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of alpha-syn by DA. We show that alpha-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of alpha-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of alpha-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of alpha-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with beta-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with alpha-syn to generate the alpha-syn aggregates implicated in Parkinson's disease.

alpha-synuclein is a small presynaptic protein (14,460 D) that is abundantly distributed in the brain. Although, its function is unknown, the aggregated form of alpha-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals that are neuro-protective. The fibrillation of alpha-synuclein was studied in relation to five different compounds found in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioflavin T assays, gel electrophoresis, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and atomic force microscopy (AFM) were utilized to monitor the rate of alpha-synuclein fibrillation and the inhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit alpha-synuclein fibril formation in a concentration-dependent manner, with nicotine being more effective. The SEC-HPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of the oligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomers with an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of the cigarette smoke components on the A53T mutant fibrillation. These results show that nicotine and hydroquinone inhibit alpha-synuclein fibrillation and stabilize soluble oligomeric forms. This information can be used to understand the molecular mechanism of the nicotine and hydroquinone action to develop therapeutic solutions for PD.

Alpha-synuclein is an intrinsically unfolded protein that can adopt a partially helical structure when it interacts with different lipid membranes. Its pathological relevance is linked to its involvement in several neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, and dementia with Lewy bodies. Typical of such ailments is the presence of alpha-synuclein aggregates in a beta-structure that can be soluble or precipitate. This review focuses on the structural knowledge acquired in recent years on the various conformations accessible to alpha-synuclein and to its pathologically relevant mutants. Furthermore, the role of the different variables of the chemical environments that govern the equilibria among the accessible conformations is also reviewed. The hypotheses that rationalize the relevance of the individual structural features and conformations for the physiological function of the protein or for its purported pathological role are described and compared.

The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human alpha-synuclein on the mean length of mature alpha-synuclein fibrils, which are associated with Parkinson's disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at approximately 15 muM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern alpha-synuclein fibril formation.

Fibrils from the Parkinson's-disease-related A53T mutant of alpha-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from beta-strand character, in addition to the rigid beta-sheet-rich core region. As in earlier studies on wild-type alpha-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in beta-sheets. Compared to fibrils from wild-type alpha-synuclein, the well-ordered beta-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of alpha-synuclein differs in both aggregation kinetics and fibril structure.

alpha-Synuclein is a neural protein that comprises the fibrillar core of Lewy bodies, a histologically defining lesion of Parkinson's disease. To investigate the role of each specific residue of the alpha-synuclein molecule in fibril formation, amino acid substitutions were introduced throughout the molecule. Incorporation of proline, especially in the region spanning residues 37-89, drastically retarded fibril formation. Substitutions with polar residues showed that the hydrophobicity of the central hydrophobic region is also important in fibrillation regulation. In the N-terminal repeated region, increasing the number of negative charges interfered with fibrillation. In contrast, single amino acid substitutions in the C-terminal acidic region of alpha-synuclein had only minimal effects on fibrillation. More than 20 different single amino acid substitutions that were sufficient to prevent fibrillation of alpha-synuclein were obtained, and most of them were impaired in both nucleation and fibril elongation. Identification of sequence determinants regulating fibrillation of amyloidogenic proteins may provide valuable information for designing peptide analog drugs to prevent protein amyloidosis.

Alpha-synuclein (alpha-Syn) fibrils are the major component of Lewy bodies that are closely associated with the pathogenesis of Parkinson's disease, but the mechanism for the fibril assembly remains poorly understood. Here we report using a combination of peptide truncation and atomic force microscopy (AFM) to elucidate the self-assembly and morphology of the alpha-Syn fibrils. The results show that protease K significantly slims the fibrils from the mean height of approximately 6.6 to approximately 4.7 nm, whereas chaotropic denaturant urea completely breaks down the fibrils into small particles. The in situ enzymatic digestion also results in thinning of the fibrils, giving rise to some nicks on the fibrils. Moreover, N- or C-terminally truncated alpha-Syn fragments assemble into thinner filaments with the heights depending on the peptide lengths. A nine-residue peptide corresponding to the homologous GAV-motif sequence can form very thin (approximately 2.2 nm) but long (>1 microm) filaments. Thus, the central sequence of alpha-Syn forms a fibrillar core by cross-beta-structure that is flanked by two flexible termini, and the orientation of the fibril growth is perpendicular to the beta-sheet structures.

There is substantial evidence which implicates alpha-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between alpha-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the alpha-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of 'small features' consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the presence of filamentous inclusions in nerve cells. These filaments are amyloid fibrils that are made of the protein alpha-synuclein, which is genetically linked to rare cases of PD and DLB. beta-Synuclein, which shares 60% identity with alpha-synuclein, is not found in the inclusions. Furthermore, while recombinant alpha-synuclein readily assembles into amyloid fibrils, beta-synuclein fails to do so. It has been suggested that this may be due to the lack in beta-synuclein of a hydrophobic region that spans residues 73-83 of alpha-synuclein. Here, fibril assembly of recombinant human alpha-synuclein, alpha-synuclein deletion mutants, beta-synuclein and beta/alpha-synuclein chimeras was assayed quantitatively by thioflavin T fluorescence and semi-quantitatively by transmission electron microscopy. Deletion of residues 73-83 from alpha-synuclein did not abolish filament formation. Furthermore, a chimera of beta-synuclein with alpha-synuclein(73-83) inserted was significantly less fibrillogenic than wild-type alpha-synuclein. These findings, together with results obtained using a number of recombinant synucleins, showed a correlation between fibrillogenesis and mean beta-strand propensity, hydrophilicity and charge of the amino acid sequences. The combination of these simple physicochemical properties with a previously described calculation of beta-strand contiguity allowed us to design mutations that changed the fibrillogenic propensity of alpha-synuclein in predictable ways.

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.

alpha-Synuclein filaments are the central component of intracytoplasmic inclusion bodies characteristic of Parkinson's disease (PD) and related disorders. Metals are the significant etiological factors in PD, and their interaction with alpha-synuclein affect dramatically the kinetics of fibrillation. Currently, we have investigated the influence of Cu(II) and Fe(III) on alpha-synuclein fibril formation. Cu(II) and Fe(III) selectively and differentially induced the formation of discrete alpha-synuclein fibrillar species. Transmission electron microscopy was used to monitor the aggregation state of alpha-synuclein (wild-type, A30P, A53T, and E46K) after 60h with stirring at 37 degrees C in the presence and absence of metal ions. Cu(II) has induced thin long network-like fibrils with the wild-type of alpha-synuclein, while the mutant, showed amorphous aggregates with no fibrillar forms. Fe(III) induced short and thick fibrils with both wild and mutant forms and were similar to alpha-synuclein fibrils incubated without metal ion. The present study illustrates the metal-specific fibril morphology, and has relevance in understanding the role of metals in neurodegeneration.

alpha-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenerative disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson's disease and Lewy body dementias, alpha-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson's diseases are linked to mutations in the gene for alpha-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins associated with a neurodegenerative disorder, this protein has now been suggested to bind copper. This finding is currently controversial. This review examines the evidence that alpha-synuclein is a copper binding protein and discusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation.

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein alpha-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164-173). Herein, we present biochemical and biophysical characterization of E46K alpha-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K alpha-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K alpha-synuclein fibrils possess a typical amyloid ultrastructure. E46K alpha-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of alpha-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type alpha-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of alpha-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT alpha-synuclein that are observed in vitro.

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.

Accumulation of chemically altered proteins is a noted characteristic of biological aging, and increasing evidence suggests a variety of deleterious cellular developments associated with senescence. Concomitantly, the "aging" of protein deposits associated with numerous neurological disorders may involve covalent modifications of their constituents. However, the link between disease-related protein aggregation and chemical alterations of its molecular constituents has yet to be established. The present study of amyloidogenic alpha-synuclein protein points to a decisive change in the biophysical behavior of growing protein aggregates with progressive photo-activity in the visible range of the electromagnetic spectrum. I hypothesize that the photo-activity induced by filament formation is governed by the same mechanism as seen for the intrinsic chromophore of 4-(p-hydroxybenzylidene)-5-imidazolinone-type in the family of green fluorescent proteins. This type of the covalent alterations is initiated concurrently with amyloid elongation and involves a complex multi-step process of chain cyclization, amino acid dehydration, and aerial oxidation. Given that different stages in filament formation yield distinct optical characteristics, the photo-activity induced by amyloidogenesis may have application in molecular biology by enabling in vivo visualization of protein aggregation and its impact on cellular function.

Different conditions, ranging from genetic mutation to post-translational modification, result in the intracellular presence of misfolded or conformationally altered proteins. These abnormal proteins tend to organize in toxic oligomeric structures often resulting in cellular death. Alterations in the function of the surveillance systems that normally repair or remove abnormal proteins are the basis of many neurodegenerative disorders. In this review, we focus on such protein conformational disorders and on the role that altered function of intracellular proteolytic systems, in particular autophagy, plays in the evolution of these diseases. Using Parkinson disease as a main example, we recapitulate the different stages of this protein conformational disorder at the cellular level and relate them with changes in the different types of autophagy. Finally, we also comment on the effect that aggravating conditions, such as oxidative stress and aging, have on the functioning of the autophagic system and its ability to cope with altered proteins.

Pyrroloquinoline quinone (PQQ) is a noncovalently bound cofactor in the bacterial oxidative metabolism of alcohols. PQQ also exists in plants and animals. Due to its inherent chemical feature, namely its free-radical scavenging properties, PQQ has been drawing attention from both the nutritional and the pharmacological viewpoint. alpha-Synuclein, a causative factor of Parkinson's disease (PD), has the propensity to oligomerize and form fibrils, and this tendency may play a crucial role in its toxicity. We show that PQQ prevents the amyloid fibril formation and aggregation of alpha-synuclein in vitro in a PQQ-concentration-dependent manner. Moreover, PQQ forms a conjugate with alpha-synuclein, and this PQQ-conjugated alpha-synuclein is also able to prevent alpha-synuclein amyloid fibril formation. This is the first study to demonstrate the characteristics of PQQ as an anti-amyloid fibril-forming reagent. Agents that prevent the formation of amyloid fibrils might allow a novel therapeutic approach to PD. Therefore, together with further pharmacological approaches, PQQ is a candidate for future anti-PD reagent compounds.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most prevalent neurodegenerative diseases of the central nervous system. These two diseases share a common feature in that a normally soluble peptide (amyloid-beta) or protein (alpha-synuclein) aggregates into an ordered fibrillar structure. As well as structural similarities observed between fibrillar aggregates related to these diseases, common pathological processes of increased oxidative injury, excitotoxicity and altered cell cycle are also evident. It was the aim of this study to identify novel interacting proteins to the amyloid-like motif and therefore identify common potential pathways between neurodegenerative diseases that share biophysical properties common to classical amyloid fibrils. Optimal ageing of recombinant proteins to form amyloid-like fibrils was determined by electron microscopy, Congo red birefringement and photo-induced cross-linking. Using pull-down assays the strongest detected interacting protein to the amyloid-like motifs of amyloid-beta, alpha-synuclein and lysozyme was identified as histone H1. The interaction with the amyloid-like motif was confirmed by techniques including surface plasmon resonance and immunohistochemistry. Histone H1 is known to be an integral part of chromatin within the nucleus, with a primary role of binding DNA that enters and exits from the nucleosome, and facilitating the shift in equilibrium of chromatin towards a more condensed form. However, phosphorylated histone H1 is predominantly present in the cytoplasm and as yet the functional significance of this translocation is unknown. This study also found that histone H1 is localised within the cytoplasm of neurons and astrocytes from areas affected by disease as well as amyloid plaques, supporting the hypothesis that histone H1 favoured binding to an ordered fibrillar motif. We conclude that the binding of histone H1 to a general amyloid-like motif indicates that histone H1 may play an important common role in diseases associated with amyloid-like fibrils.

alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.

DJ-1 has been reported to have chaperone activity by preventing the aggregation of some proteins, and by structural analogy to Hsp31. The L166P mutation has been linked to a familial early onset form of Parkinson's disease (PD). Since the aggregation of alpha-synuclein is believed to be a critical step in the etiology of PD, we have investigated the interaction of wild-type DJ-1 and its oxidized forms with alpha-synuclein. Native (unoxidized) DJ-1 did not inhibit alpha-synuclein fibrillation, and no evidence for stable interactions between alpha-synuclein and native DJ-1 was observed. However, DJ-1 is very susceptible to oxidation by the addition of two oxygen atoms to form the sulfinic acid of Cys106 (2O DJ-1) (no 1O oxidized state is detectable). 2O DJ-1 was readily prepared by the addition of H(2)O(2) at concentrations up to a 20-fold molar excess. The oxidation of Cys106 to the sulfinic acid had minimal effect on the structural properties of DJ-1. However, 2O DJ-1 was very effective in preventing the fibrillation of alpha-synuclein, and only this form of DJ-1 appears to have significant anti-aggregation properties against alpha-synuclein. Further oxidation of DJ-1 leads to loss of some secondary structure, and to loss of the ability to inhibit alpha-synuclein fibrillation. Our observations confirm the suggestion that DJ-1 may act as an oxidative-stress-induced chaperone to prevent alpha-synuclein fibrillation. Since oxidative stress has been associated with PD, this observation may explain why mutations of DJ-1 could be a contributing factor in PD, and also indicates that excess oxidative stress could also lead to enhanced alpha-synuclein aggregation and hence PD.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.

In this report, we applied in situ atomic force microscopy (AFM) to study the dynamic process of disassembly-assembly of alpha-synuclein (alpha-Syn) fibrils in different solutions. Most of the mica-adsorbed alpha-Syn fibrils disassemble into small particles step-by-step on the mica surface in diluted solutions, yet a few short fibrils still extend to form longer fibrils. This process usually started randomly at the center of the long fibrils, which progressively disassemble into short fragments and small protein particles of varying size. Compared to disassembly, assembly happened infrequently when the protein concentration was low. It was observed directly by AFM that the chaotropic agent guanidinium chloride rapidly breaks the long alpha-Syn fibrils.

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.

The deposition of alpha-syn (alpha-synuclein) fibrils in Lewy bodies is a characteristic feature of individuals with neurodegenerative disorders. A peptide comprising the central residues 71-82 of alpha-syn [alpha-syn(71-82)] is capable of forming beta-sheet-rich, amyloid-like fibrils with similar morphologies to fibrils of the full-length protein, providing a useful model of pathogenic alpha-syn fibrils that is suitable for detailed structural analysis. We have studied the morphology and gross structural features of alpha-syn(71-82) fibrils formed under different conditions in order to obtain reliable conditions for producing fibrils for further structural investigations. The results indicate that the rate of aggregation and the morphology of the fibrils formed are sensitive to pH and temperature.