FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

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.

Inhibition of α-Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends.

Misfolding of the protein α-synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh-ß) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh-ß concentrations (IC50 =850 nm) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.

Modification of C Terminus Provides New Insights into the Mechanism of α-Synuclein Aggregation.

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).

Environmentally sensitive probes for monitoring protein-membrane interactions at nanomolar concentrations.

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.

A four-amino acid linker between repeats in the α-synuclein sequence is important for fibril formation.

α-Synuclein is a 140-amino acid protein that can switch conformation among intrinsically disordered in solution, helical on a membrane, and ß-sheet in amyloid fibrils. Using the fluorescence of single-tryptophan mutants, we determined the immersion of different regions of the protein into lipid membranes. Our results suggest the presence of a flexible break close to residues 52-55 between two helical domains. The four-amino acid linker is not necessary for membrane binding but is important for fibril formation. A deletion mutant lacking this linker aggregates extremely slowly and slightly inhibits wild-type aggregation, possibly by blocking the growing ends of fibrils.

Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-ß-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms.

Dual-fluorescence L-amino acid reports insertion and orientation of melittin peptide in cell membranes.

Monitoring insertion and orientation of peptides in situ on cell membranes remains a challenge. To this end, we synthesized an l-amino acid (AFaa) containing a dual-fluorescence dye of the 3-hydroxyflavone family, as a side chain. In contrast to other labeling approaches using a flexible linker, the AFaa fluorophore, introduced by solid phase synthesis into desired position of a peptide, is attached closely to its backbone with well-defined orientation, and, therefore, could reflect its localization in the membrane. This concept was validated by replacing the leucine-9 (L9) and tryptophan-19 (W19) residues by AFaa in melittin, a well-studied membrane-active peptide. Due to high sensitivity of AFaa dual emission to the environment polarity, we detected a much deeper insertion of L9 peptide position into the bilayer, compared to the W19 position. Moreover, using fluorescence microscopy with a polarized light excitation, we found different orientation of AFaa at L9 and W19 positions of melittin in the bilayers of giant vesicles and cellular membranes. These results suggested that in the natural membranes, similarly to the model lipid bilayers, melittin is preferentially oriented parallel to the membrane surface. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides.

Dopamine-Induced Oligomers of α-Synuclein Inhibit Amyloid Fibril Growth and Show No Toxicity.

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.

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe.

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.

Two-color fluorescent l-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes.

Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.

Quantification of local hydration at the surface of biomolecules using dual-fluorescence labels.

By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that W(A) values for the label at the peptide N-terminus are lower (0.63-0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (W(A) = 0-0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure.

Improved hydration-sensitive dual-fluorescence labels for monitoring peptide-nucleic acid interactions.

Environmentally sensitive labels constitute a new, attractive tool for monitoring biomolecular interactions. 3-Hydroxychromone derivatives are of particular interest because they undergo excited-state intramolecular proton transfer (ESIPT) showing dual emission highly sensitive to environmental hydration. To overcome the drawbacks of the previously developed label for sensing protein-DNA interactions based on 2-furanyl-3-hydroxychromone (FC), a series of hydration-sensitive labels based on 3-hydroxy-4'-methoxyflavone have been synthesized. As compared to FC, the new labels display higher sensitivity of the ratio of their two emission bands (N*/T*) to solvent polarity and H-bond donor ability, as well as higher fluorescence quantum yields in water. Moreover, they show higher pK(a) values of their 3-hydroxyl group, allowing their application at neutral pH without interference of anionic forms. To illustrate the applications of these labels, we covalently coupled them to the N-terminus of the Tat(44-61) peptide that corresponds to the basic domain of the HIV-1 Tat protein. This coupling did not modify the nucleic acid chaperone properties of the peptide. Binding of oligonucleotides of varying length, sequence, and strandedness to the labeled peptides induced dramatic change in the N*/T* ratio of their two emission bands. This change indicated that the level of probe hydration in the peptide/oligonucleotide complexes decreases in the following order: short ssDNAs ≫ long ssDNAs > DNA hairpins > dsDNAs. The level of probe hydration was related to the ability of the probe to stack with the DNA bases or base pairs in the various complexes. The changes in the N*/T* ratio upon interaction of the labeled Tat peptides with DNA were about 3-fold larger with the new probes as compared to the parent FC label, in line with the higher sensitivity of the new probes to the environment. One of these labels, presenting the most compact geometry, showed the highest sensitivity, probably due to its optimal stacking with the DNA bases. Thus, the new hydration-sensitive labels appear as improved highly sensitive tools to site-selectively monitor the binding of peptides to oligonucleotides and nucleic acids.

Sensing peptide-oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein.

We present a new methodology for site-specific sensing of peptide-oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide-ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide-ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide-ODN interactions.

Influence of Lipid Membranes on α-Synuclein Aggregation.

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.

Rationally Designed Protein-Based Inhibitor of α-Synuclein Fibrillization in Cells.

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.

Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells.

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.

A peptide-based fluorescent ratiometric sensor for quantitative detection of proteins.

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4(Q34S), with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3 HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4(Q34S) to labeled peptide. The detection limit of scFv1F4(Q34S) was 15 nM. In a more complex medium (100 microg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.

Structural Optimization of Inhibitors of α-Synuclein Fibril Growth: Affinity to the Fibril End as a Crucial Factor.

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 ß-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.

Reversible spatial and temporal control of lipid signaling.

Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.

Dual-fluorescence probe of environment basicity (hydrogen bond accepting ability) displaying no sensitivity to polarity.

3-Hydroxyquinolones (3HQs) are a new class of water soluble dual fluorescence probes that can monitor both polarity and basicity (H-bond accepting ability) parameters. Both parameters play an important role in proteins and lipid membranes. Nevertheless, no method exists actually to measure the basicity parameter separately from the polarity. To achieve this aim, we synthesized 2-benzofuryl-3-hydroxy-4(1H)-quinolone (3HQ-Bf) and characterized its photophysical properties by UV, steady-state and time-resolved fluorescence spectroscopy. Due to its extended conjugation and totally planar conformation, 3HQ-Bf is characterized by a high fluorescence quantum yield. In solution, this dye shows an excited state intramolecular proton transfer (ESIPT) reaction resulting in two tautomer bands in the emission spectra. The ESIPT reaction can be considered as irreversible and is governed by rate constants from 0.6 to 8 x 10(9) s(-1), depending on the solvent. The analysis of the spectral properties of 3HQ-Bf in a series of organic solvents revealed a marginal sensitivity to the solvent polarity, but an exquisite sensitivity to solvent basicity, as shown by the linear dependence of the logarithm of the emission bands intensity ratio, log(I(N*)/I(T*)), as well as the absorption or emission maxima wavenumbers as a function of the solvent basicity parameter. This probe may find useful applications through coupling to a protein ligand, for characterizing the H-bond acceptor ability at the ligand binding site as well as for studying the basicity changes of lipid membranes during their chemo- and thermotropic conversions.