Conformation Of Alpha-synuclein Research Articles

Structural and Aggregation Properties of Alpha-Synuclein Linked to Phospholipase A2 Action.

Alpha-synuclein is a protein involved in the pathogenesis of Parkinson's disease. In vitro observations have shown that specific brain-enriched polyunsaturated fatty acids, such as arachidonic acid, can give rise to a conformational change in alpha-synuclein and ultimately induce its fibrillation. Arachidonic acid is released by phospholipase A2 activity and clinical observations have shown a link between mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. It is unknown how phospholipase A2-driven release of arachidonic acid can affect the conformation of alphasynuclein. The main objective of this study was to investigate if phospholipase A2-induced release of arachidonic acid can induce changes in conformation and aggregation state of alpha-synuclein. Recombinant human alpha-synuclein was expressed and isolated and incubated in the presence of phosphatidylcholine and phosphatidylserine (PC/PS) containing liposomes. The release of free fatty acids from PC/PS liposomes by bee venom phospholipase A2 was measured with the fluorescent probe acrylodated intestinal fatty acid-binding protein (ADIFAB) and radioactive labelling by preparing liposomes in the presence of L- 3-phosphatidylcholine, 1-stearyl-2[1-14C] arachidonoyl. The effect of free fatty acid release on the conformation of alpha-synuclein was assayed by far-UV circular dichroism and resistance against V8 protease-induced limited proteolysis. Aggregation of alpha-synuclein upon exposure to phospholipase A2-induced action on PC/PS liposomes was measured using thioflavin T fluorescence, SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. RAW264.7 cells were transiently transfected with human alpha-synuclein and release of arachidonic acid was quantified using radiolabeling and liquid scintillation counting. Phospholipase A2 is capable of releasing arachidonic acid from biomimetic phospholipid membranes. Exposure of alpha-synuclein to phospholipase A2-induced release of arachidonic acid from PC/PS liposomes induces a conformational transition of the protein and leads to partial resistance against proteolytic cleavage by V8 protease. Prolonged incubation of alpha-synuclein with arachidonic acid, derived from PC/PS liposomes by phospholipase A2 leads to aggregate formation. In line with this, transiently transfected RAW264.7 cells with alpha-synuclein showed arachidonic acid release and punctate alpha-synuclein staining upon phospholipase A2 activation. The ability of arachidonic acid to drive alpha-synuclein to aggregate was independent of its oxidation state. We present data that suggest a biological context for the previously reported clinical observation that linked mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. Release of arachidonic acid, independent of its oxidation state, through activation of phospholipase A2-driven hydrolysis of phospholipid membranes, leads to the structural transition and aggregation of alpha-synuclein.

The Conformation of Alpha-Synuclein on the Natural Membrane Mimics Inner Mitochondrial Membrane (IMM) and Neuronal Plasma Membrane (NPM) Revisited by Bayesian Analysis of Double Electron Electron Resonance (DEER) Distance Distributions

The Conformation of Alpha-Synuclein on the Natural Membrane Mimics Inner Mitochondrial Membrane (IMM) and Neuronal Plasma Membrane (NPM) Revisited by Bayesian Analysis of Double Electron Electron Resonance (DEER) Distance Distributions

Temperature, surface morphology and biochemical cues: A combined approach to influence the molecular conformation of Alpha-synuclein

Here we report an investigation on conformational changes of Alpha-synuclein (α-syn) and one of its mutations (E46K), whose fibrillar assemblies in neural cells are the molecular hallmark of Parkinsons' disease. By varying the environmental conditions of the two peptides in terms of temperature (21–60°C), the presence of lipid systems (in homogeneous fluid phase or presenting raft-like domains) and nanostructured surfaces, we observed significant secondary structure alterations in both proteins. In order to probe such transitions, we exploited synchrotron μFTIR directly on solid ring-like residues formed immediately upon evaporation. We concluded that high temperatures and the presence of a lipid fluid phase induce a transition from α-helical materials to β-sheet ones (the characteristic conformation of α-syn fibrils) on pristine flat substrates. The presence of lipid rafts hindered this transition. On the other hand, the inclination to form β-sheet phases in the presence of high temperatures or lipid systems was sensitively lowered by drying the proteins on top of nanostructured highly hydrophilic supports which allow a more homogeneous distribution of the analyte throughout the residue. These results and the presented protocol can indeed pave the basis for the structural characterization of this controversially debated protein and of other neurodegenerative peptides.

Dopamine-Induced Conformational Changes in Alpha-Synuclein

BackgroundOligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson's disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7].Methodology/Principal FindingsHere, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species.Conclusion/SignificanceOur results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinson's disease.

Effect of metals on herbicides–α‐synuclein association: A possible factor in neurodegenerative disease studied by capillary electrophoresis

The aggregation of alpha-synuclein in the dopaminergic neurons of the substantia nigra is a critical step in the Parkinson's disease (PD). The etiology of the disease is unknown but recent epidemiological and experimental studies have renewed interest in the hypothesis that environmental factors, especially herbicides and metals, have a role on the pathogenesis of PD. For the first time, the association constants of alpha-synuclein with five herbicides have been calculated using a capillary electrophoresis (CE) method. In addition, the effect of a number of metals on this binding has been investigated. It appears that the herbicides preferentially bind to a partially folded intermediate conformation of alpha-synuclein induced by manganese, aluminium, cadmium, copper and zinc. Then, metal increases the synuclein-herbicide association. However, this study shows contrasting actions with the antibiotic rifampicin and magnesium addition leading to a decrease of the alpha-synuclein-herbicide interaction even if other metals are present in the bulk solvent. Considering epidemiological studies, all these results suggest an underlying molecular basis for PD and related body diseases.

Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: comparison between Gallyas-Braak and Campbell-Switzer methods

Lewy bodies (LBs) of idiopathic Parkinson's disease and glial cytoplasmic inclusions (GCIs) of multiple system atrophy are pathological deposits both composed of phosphorylated alpha-synuclein woven into different filaments. Although both LBs and GCIs are considered to be hallmarks for each independent synucleinopathy, until now they could not be clearly distinguished on the basis of their biochemical or immunohistochemical features. We have examined possible differences in their argyrophilic features and their relation to synuclein-like or ubiquitin-like immunoreactivity (IR). Pairs of mirror sections from different brain areas were triple-fluorolabeled with an anti-alpha-synuclein antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that labels fibrillary structures such as Lewy bodies or neurofibrillary tangles. One of the paired sections was subsequently stained using the Campbell-Switzer method (CS), and the other by the Gallyas-Braak method (GB). By comparing of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either CS or GB, five different profiles of each structure could be determined: alpha-synuclein-like IR, ubiquitin-like IR, affinity to TR, argyrophilia with CS or GB. GCIs exhibited argyrophilia with both CS and GB but lacked affinity to TR. In contrast, LBs exhibited argyrophilia with CS but not with GB and some affinity to TR. These disease-specific profiles of argyrophilia were consistent, and were not influenced by areas or cases examined. Although immunohistochemical features of LBs and GCIs were similar in exhibiting IR for alpha-synuclein and ubiquitin, the contrast in their argyrophilic profiles may indicate possible differences in the molecular composition or conformation of alpha-synuclein. Even though these empirical differences still remain to be explained, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

The Association of α-Synuclein with Membranes Affects Bilayer Structure, Stability, and Fibril Formation

The aggregation of alpha-synuclein is believed to be a critical factor in the etiology of Parkinson's disease. alpha-Synuclein is an abundant neuronal protein of unknown function, which is enriched in the presynaptic terminals of neurons. Although alpha-synuclein is found predominantly in the cytosolic fractions, membrane-bound alpha-synuclein has been suggested to play an important role in fibril formation. The effects of alpha-synuclein on lipid bilayers of different compositions were determined using fluorescent environment-specific probes located at various depths. alpha-Synuclein-membrane interactions were found to affect both protein and membrane properties. Our results indicate that in addition to electrostatic interactions, hydrophobic interactions are important in the association of the protein with the bilayer, and lead to disruption of the membrane. The latter was observed by atomic force microscopy and fluorescent dye leakage from vesicles. The kinetics of alpha-synuclein fibril formation were significantly affected by the protein association and subsequent membrane disruption, and reflected the conformation of alpha-synuclein. The ability of alpha-synuclein to disrupt membranes correlated with the binding affinity of alpha-synuclein for the particular membrane composition, and to the induced helical conformation of alpha-synuclein. Protofibrillar or fibrillar alpha-synuclein caused a much more rapid destruction of the membrane than soluble monomeric alpha-synuclein, indicating that protofibrils (oligomers) or fibrils are likely to be significantly neurotoxic.

A Broken α-Helix in Folded α-Synuclein

alpha-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. alpha-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, alpha-synuclein is natively unstructured but assumes an alpha-helical conformation upon binding to phospholipid membranes. We now show that this conformation of alpha-synuclein consists of two alpha-helical regions that are interrupted by a short break. The structural organization of the alpha-helices of alpha-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.

Magnesium Inhibits Spontaneous and Iron-induced Aggregation of α-Synuclein

Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to alpha-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in alpha-synuclein as indicators of metal-related conformational changes in alpha-synuclein, we show that iron and magnesium both interact with alpha-synuclein. alpha-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of alpha-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits alpha-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases alpha-synuclein aggregation, while magnesium at concentrations >0.75 mm inhibits the aggregation of alpha-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of alpha-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation.

Interaction of α‐synuclein and synphilin‐1: effect of Parkinson's disease‐associated mutations

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.

Membrane Association and Protein Conformation of α-Synuclein in Intact Neurons: EFFECT OF PARKINSON′S DISEASE-LINKED MUTATIONS

Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.