Parkinson's disease is characterized by the selective death of dopaminergic neurons in the midbrain and accumulation of amyloid fibrils composed of α-synuclein (αSyn). Current treatment involves approaches that compensate the death of dopaminergic neurons by increasing the dopamine levels in remaining cells. However, dopamine can interact with αSyn and produce oligomeric species which were reported to be toxic in many models. We studied formation of dopamine-induced αSyn oligomers and their effect on the αSyn aggregation. Using the Thioflavin T kinetic assay, we have shown that small oligomers efficiently inhibit αSyn fibrillization by binding to fibril ends and blocking the elongation. Moreover, all the fractions of oligomer species proved to be nontoxic in the differentiated SH-SY5Y cell model and showed negligible neurotoxicity on isolated rat synaptosomes. The observed inhibition is an important insight in understanding of dopamine-enhancing therapy on Parkinson's disease progression and explains the absence of pathology enhancement.
- MeSH
- alfa-synuklein metabolismus MeSH
- amyloid metabolismus MeSH
- dopamin chemie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- neuroblastom * MeSH
- Parkinsonova nemoc * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.
- MeSH
- alfa-synuklein analýza antagonisté a inhibitory metabolismus MeSH
- benzodioxoly farmakologie MeSH
- elektroporace metody MeSH
- HeLa buňky MeSH
- intracelulární tekutina chemie účinky léků metabolismus MeSH
- lidé MeSH
- proteinové agregáty účinky léků fyziologie MeSH
- pyrazoly farmakologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- viabilita buněk účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Misfolding of the neuronal protein α-synuclein (αSyn) into amyloid fibrils is involved in the development of Parkinson's disease (PD), and inhibition of this process is considered to be a promising therapeutic approach. In this work, we engineered protein inhibitors that bind to fibrils with higher affinity than the monomeric αSyn. They were developed based on the recent structural data of the αSyn fibrils and were shown to prevent fibril elongation upon binding to fibril ends. These inhibitors are highly selective to the misfolded αSyn, nontoxic, and active in cytosol in small concentrations. The best-performing inhibitor shows IC50 ∼10 nM in a cell-based assay, which corresponds to the ∼1:60 molar ratio to αSyn. It can suppress the formation of αSyn aggregates in cells that can be potentially used to slow down the spreading of the pathological aggregates from cell to cell during the course of the PD.
- MeSH
- alfa-synuklein antagonisté a inhibitory genetika metabolismus MeSH
- amyloid metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie MeSH
- kinetika MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- Parkinsonova nemoc metabolismus patologie MeSH
- peptidy chemie metabolismus MeSH
- proteinové agregáty MeSH
- racionální návrh léčiv * MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plaques of amyloid fibrils composed of neuronal protein α-synuclein are one of the hallmarks of Parkinson's disease, and their selective imaging is crucial to study the mechanism of its pathogenesis. However, the existing fluorescent probes for amyloids are efficient only in solution and tissue systems, and they are not selective enough for the visualization of amyloid fibrils in living cells. In this study, we present two molecular rotor-based probes RB1 and RB2. These thiazolium probes show affinity to α-synuclein fibrils and turn-on fluorescence response upon interactions. Because of its extended π-conjugation and high rotational degree of freedom, RB1 exhibits a 76 nm red-shift of absorption maxima and 112-fold fluorescence enhancement upon binding to amyloid fibrils. Owing to its strong binding affinity to α-synuclein fibrils, RB1 can selectively stain them in the cytoplasm of living HeLa and SH-SY5Y cells with high optical contrast. RB1 is a cell-permeable and noncytotoxic probe. Taken together, we have demonstrated that RB1 is an amyloid probe with an outstanding absorption red-shift that can be used for intracellular imaging of α-synuclein fibrils.
- MeSH
- alfa-synuklein * MeSH
- amyloid MeSH
- fluorescenční barviva MeSH
- fluorescenční spektrometrie MeSH
- lidé MeSH
- Parkinsonova nemoc * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
α-Synuclein is a neuronal protein involved in synaptic vesicle trafficking. During the course of Parkinson's disease, it aggregates, forming amyloid fibrils that accumulate in the midbrain. This pathological fibrillization process is strongly modulated by physiological interactions of α-synuclein with lipid membranes. However, the detailed mechanism of this effect remains unclear. In this work, we used environment-sensitive fluorescent dyes to study the influence of model lipid membranes on the kinetics of α-synuclein fibrillization. We observed that formation of the fibrils from α-synuclein monomers is strongly delayed even by small amounts of lipids. Furthermore, we found that membrane-bound α-synuclein monomers are not involved in fibril elongation. Hence, presence of lipids slows down fibril growth proportionally to the fraction of membrane-bound protein.
- MeSH
- alfa-synuklein * MeSH
- amyloid MeSH
- kinetika MeSH
- lidé MeSH
- lipidy MeSH
- Parkinsonova nemoc * MeSH
- patologická konformace proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Misfolding of the neuronal protein α-synuclein into amyloid fibrils is a pathological hallmark of Parkinson's disease, a neurodegenerative disorder that has no cure. Inhibition of the fibril growth is considered a promising therapeutic approach. However, the majority of the existing inhibitors are either unspecific or work at high micromolar concentrations. Earlier, we created a protein-based inhibitor of α-synuclein fibril growth that consists of an α-synuclein moiety and a bulky group. It specifically binds to α-synuclein fibril ends and blocks them by creating steric hindrance to subsequent monomer binding. RESULTS: In this work, we prepared a series of inhibitors with modified α-synuclein moieties and bulky groups of different structure, size, and position. We studied the structure-activity relationship of these inhibitors and optimized them by improving affinity to the fibril end and blocking efficiency. The inhibitors were tested in a Thioflavin T-based kinetic assay, and their affinity to the fibril ends was measured by fluorescence anisotropy. We showed that decrease in electrostatic repulsion between inhibitor and fibril end improved the inhibitor efficiency. Inhibitors with rigid β-sheet-rich bulky groups bind to fibril ends stronger than monomeric α-synuclein and therefore have a high inhibition efficiency, showing a linear correlation between Kd and IC50. SIGNIFICANCE: We determined which properties of inhibitor molecules are the most important for good performance and found that the inhibitor affinity to the fibril end is a key feature that determines its inhibition efficiency. Applying this knowledge, we improved existing inhibitors and reached IC50 value of 300 nM.
- MeSH
- alfa-synuklein chemie metabolismus MeSH
- amyloid chemie metabolismus MeSH
- benzothiazoly chemie metabolismus MeSH
- fluorescenční polarizace MeSH
- kinetika MeSH
- lidé MeSH
- proteinové agregáty MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aggregation of the neuronal protein α-synuclein into amyloid fibrils plays a central role in the development of Parkinson's disease. Growth of fibrils can be suppressed by blocking fibril ends from their interaction with monomeric proteins. In this work, we constructed inhibitors that bind to the ends of α-synuclein amyloid fibrils with very high affinity. They are based on synthetic α-synuclein dimers and interact with fibrils via two monomeric subunits adopting conformation that efficiently blocks fibril elongation. By tuning the charge of dimers, we further enhanced the binding affinity and prepared a construct that inhibits fibril elongation at nanomolar concentration (IC50 ≈ 20 nM). To the best of our knowledge, it is the most efficient inhibitor of α-synuclein fibrillization.
- MeSH
- alfa-synuklein antagonisté a inhibitory genetika metabolismus MeSH
- amyloid antagonisté a inhibitory chemie metabolismus MeSH
- cirkulární dichroismus MeSH
- disulfidy chemie MeSH
- látky ovlivňující centrální nervový systém chemie farmakologie MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with the protein concentration. For the development of efficient inhibitors of α-synuclein aggregation, it is important to know the critical concentration of aggregation (the concentration of monomeric protein, below which the protein does not aggregate). METHODS: We performed in vitro aggregation studies of α-synuclein at low concentrations (0.11-20 μM). Aggregation kinetics was measured by ThT fluorescence. Obtained aggregates were characterized using CD-spectroscopy, fluorescent spectroscopy, dynamic light scattering and AFM imaging. RESULTS: Monomeric α-synuclein at concentrations 0.45 μM and above was able to bind to fibril ends resulting in fibril growth. At the protein concentrations below 0.4 μM, monomers did not fibrillize, and fibrils disaggregated. In the absence of seeds, fibrils were formed only at monomer concentrations higher than 10 μM. At low micromolar concentrations, we observed formation of prefibrillar amyloid aggregates, which are able to induce fibril formation in α-synuclein solutions of high concentrations. CONCLUSIONS: The critical concentration of α-synuclein fibril growth is ~0.4 μM. Prefibrillar amyloid aggregates appear at concentrations between 0.45 and 3 μM and are an intermediate state between monomers and fibrils. Although morphologically different from fibrils, prefibrillar aggregates have similar properties to those of fibrils. GENERAL SIGNIFICANCE: We determined the critical concentration of α-synuclein fibril growth. We showed that fibrils can grow at much lower monomer concentrations than that required for de novo fibril formation. We characterized a prefibrillar intermediate species formed upon aggregation of α-synuclein at low micromolar concentration.
Aggregation of neuronal protein α-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of α-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to α-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such α-synuclein sequence extension on α-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of α-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of α-synuclein. To address these questions, we prepared mutants of α-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type α-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of α-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of α-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).
Solvatochromic probes are suitable tools for quantitative characterization of protein-membrane interactions. Based on diverse fluorophores these probes have different fluorescent properties and therefore demonstrate different responses when applied for sensing the interactions of biomolecules. Surprisingly, to the best of our knowledge, no systematic comparison of the sensitivities of solvatochromic dyes for monitoring protein-membrane interactions was described. Hence, a rational choice of an optimal environmentally sensitive probe for such experiments is usually not a straightforward task. In this work we developed a series of thiol-reactive fluorescent probes based on the fluorophores with high sensitivity to their environment and compared them with two widely used DNS and DMN probes. We investigated the responses of these probes to the interaction of probe-labeled presynaptic protein α-synuclein with lipid membranes. We observed that newly synthesized probes based on fluorene and chromone dyes, which combine the strongest brightness and significant changes of fluorescence intensity, demonstrated the highest sensitivity to interaction of α-synuclein with lipid membranes. They are especially beneficial for sensing in scattering media such as solutions of lipid vesicles. We show that the described probes permit quantitative measurements of α-synuclein binding to lipid membranes at low nanomolar concentrations. We developed a detailed protocol for measuring Kd and binding stoichiometry for interaction of soluble peripheral proteins with membranes based on the response of the environmentally sensitive fluorescent probes. We applied this protocol for quantification of the affinity of α-synuclein to anionic membranes and found that it is substantially higher than it was earlier reported.
