Heterotypic liquid-liquid phase separation of tau and α-synuclein: Implications for overlapping neuropathologies.

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

Modulation of α-synuclein phase separation by biomolecules.

Liquid-liquid phase separation (LLPS) is currently recognized as a common mechanism involved in the regulation of a number of cellular functions. On the other hand, aberrant phase separation has been linked to the biogenesis of several neurodegenerative disorders since many proteins that undergo LLPS are also found in pathological aggregates. The formation of mixed protein coacervates may constitute a risk factor in overlapping neuropathologies, such as Parkinson's (PD) and Alzheimer's (AD) diseases. In this work, we evaluated the homotypic and heterotypic phase behaviour of the PD-related protein α-synuclein (AS) in the presence of the biologically relevant molecules ATP, polyamines, and the AD-related protein Tau. We found that AS exhibits a low propensity to form homotypic liquid droplets, yet phase separates into liquid-like or solid-like phases depending on the interacting biomolecule. We further demonstrated the synergistic droplet formation of AS and Tau providing support for a mechanism in which mixed condensates might contribute to the biogenesis of AS/Tau pathologies.

Influence of the macromolecular crowder alginate in the fibrillar organization of the functional amyloid FapC from Pseudomonas aeruginosa.

Bacterial biofilms are an alternative lifestyle in which communities of bacteria are embedded in an extracellular matrix manly composed by polysaccharides, nucleic acids and proteins, being the hallmark of bacterial survival in a variety of ecological niches. Amyloid fibrils are one of the proteinaceous components of such extracellular crowded environments. FapC is the main component of the functional amyloid recently discovered in Pseudomonas species, including the opportunistic pathogen P. aeruginosa, which is a major cause of nosocomial infections and contamination of medical devices. Considering that several functional roles have been attributed to this bacterial amyloid, FapC emerged as a novel target to control Pseudomonas biofilm formation and to design new treatments against chronic infections. In this study, we used complementary biophysical techniques to evaluate conformational signatures of FapC amyloids formed in the presence of alginate, the major exopolysaccharide associated with the mucoid phenotype of P. aeruginosa strains isolated from cystic fibrosis patients. We found that the this naturally occurring macromolecular crowder leads to morphological similar yet polymorphic FapC fibrils, highlighting the importance of considering the complexity of the extracellular matrix in order to improve our understanding of microbial functional amyloids.

Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein.

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel ß-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel ß-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel ß-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

Structural insights into amyloid oligomers of the Parkinson disease-related protein α-synuclein.

The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the ß-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations.

Structural characterization of heparin-induced glyceraldehyde-3-phosphate dehydrogenase protofibrils preventing α-synuclein oligomeric species toxicity.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

Imaging nanometer-sized α-synuclein aggregates by superresolution fluorescence localization microscopy.

The morphological features of α-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.

Small molecule fluorescent probes for the detection of amyloid self-assembly in vitro and in vivo.

The misfolding and aggregation of amyloidogenic polypeptides are characteristics of many neurodegenerative syndromes including Alzheimer's and Parkinson's disease. There is a major interest in the availability of amyloid-specific probes that exhibit fluorescence properties, for its use as reporters of protein aggregation in spectroscopy and microscopy methodologies. In this review, we intend to provide an overview of novel fluorescence-based probes and procedures applied for addressing fundamental aspects of amyloid self-assembly in vitro and in vivo. We highlight the utilization in vitro of several small-molecule fluorescent probes as extrinsic and site-specific reporters of amyloid formation, including single-molecule determinations. Detection of amyloid self-assembly employing compounds such as JC-1, DCVJ, ANS derivatives and luminescent conjugated polymers, as well as site-specific probes such as pyrene and ESIPT is discussed. We further review novel fluorescent probes developed for the non-invasive optical imaging of protein aggregates in vivo, including BTA-1, Methoxy-X04, NIAD-4 and CRANAD-2. Availability of increasingly versatile amyloid-specific fluorescent probes is having a very positive impact in the drug discovery and diagnostics fields.

A triple-emission fluorescent probe reveals distinctive amyloid fibrillar polymorphism of wild-type alpha-synuclein and its familial Parkinson's disease mutants.

Intracytoplasmic neuronal deposits containing amyloid fibrils of the 140-amino acid presynaptic protein alpha-synuclein (AS) are the hallmark of Parkinson's (PD) disease and related neurodegenerative disorders. Three point mutations (A53T, A30P, and E46K) are linked to early onset PD. Compared to the wild-type (WT) protein, the mutants aggregate faster in vitro, but their fibrillar products are quite similar. Using the extrinsic multiple-emission probe 4'-(diethylamino)-3-hydroxyflavone (FE), we demonstrate unique and distinct spectroscopic signatures for the amyloid fibrils formed by the WT and mutant AS, presumably indicative of subtle differences in supramolecular structure. The two well-separated emission bands of the FE probe originate from a proton transfer reaction in the excited state. The ratiometric response constitutes a sensitive, tunable reporter of microenvironmental properties such as polarity and hydrogen bonding. The very distinctive fluorescence spectra of the FE probe bound to the four AS variants reflect different tautomeric equilibria in the excited state and the existence of at least two different binding sites in the fibrils for the dye. Deconvolution of the two-dimensional excitation-emission spectra leads to estimations of different local dielectric constants and extents of hydration characteristic of the proteins. The sensitivity of such a simple external probe to conformational alterations induced by point mutations is unprecedented and provides new insight into key phenomena related to amyloid fibrils: plasticity, polymorphism, propagation of structural features, and structure-function relationships underlying toxicity.

Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein.

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.

Fluorescent N-arylaminonaphthalene sulfonate probes for amyloid aggregation of alpha-synuclein.

The deposition of fibrillar structures (amyloids) is characteristic of pathological conditions including Alzheimer's and Parkinson's diseases. The detection of protein deposits and the evaluation of their kinetics of aggregation are generally based on fluorescent probes such as thioflavin T and Congo red. In a search for improved fluorescence tools for studying amyloid formation, we explored the ability of N-arylaminonaphthalene sulfonate (NAS) derivatives to act as noncovalent probes of alpha-synuclein (AS) fibrillation, a process linked to Parkinson's disease and other neurodegenerative disorders. The compounds bound to fibrillar AS with micromolar K(d)s, and exhibited fluorescence enhancement, hyperchromism, and high anisotropy. We conclude that the probes experience a hydrophobic environment and/or restricted motion in a polar region. Time- and spectrally resolved emission intensity and anisotropy provided further information regarding structural features of the protein and the dynamics of solvent relaxation. The steady-state and time-resolved parameters changed during the course of aggregation. Compared with thioflavin T, NAS derivatives constitute more sensitive and versatile probes for AS aggregation, and in the case of bis-NAS detect oligomeric as well as fibrillar species. They can function in convenient, continuous assays, thereby providing useful tools for studying the mechanisms of amyloid formation and for high-throughput screening of factors inhibiting and/or reversing protein aggregation in neurodegenerative diseases.

Differential scanning calorimetry as a tool to estimate binding parameters in multiligand binding proteins.

The stability of proteins and their interactions with other molecules is a topic of special interest in biochemistry because many cellular processes depend on that. New methods and approaches are constantly developed to elucidate the energetics of biomolecular recognition. In this sense, the application of the theory of macromolecular unfolding linked to ligand binding to differential scanning calorimetry (DSC) has proved to be a useful tool to simultaneously characterize the energetics of unfolding and binding. Although the general theory is well known, the applicability of DSC to study the interaction of biomolecules is not common. In the current work, we estimated the binding parameters of 8-anilinonaphthalene-1-sulfonic acid to human serum albumin using DSC. This model system was chosen due to both the complex stoichiometry and the moderate binding constants. From DSC curves acquired at different ligand concentrations, we obtained the number of bound ligands, the binding constants, and the binding enthalpy for each independent binding site. Compared with those parameters determined by titration calorimetry, the results highlight the potentiality of DSC to estimate binding parameters in multiligand binding proteins.

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction.

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.

Conformational flexibility of avidin: the influence of biotin binding.

Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.

Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.

The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.