Plant polyphenols inhibit functional amyloid and biofilm formation in <i>Pseudomonas</i> strains by directing monomers to off-pathway oligomers

Self-assembly of proteins to &beta;-sheet rich amyloid fibrils is commonly observed in various neurodegenerative diseases. However, amyloid also occurs in the extracellular matrix of bacterial biofilm, which protects bacteria from environmental stress and antibiotics. Many Pseudomonas strains produce functional amyloid where the main component is the highly fibrillation-prone protein FapC. FapC fibrillation may be inhibited by small molecules such as plant polyphenols, which are already known to inhibit formation of pathogenic amyloid, but the mechanism and biological impact of inhibition is unclear. Here, we elucidate how polyphenols modify the self-assembly of functional amyloid, with particular focus on epigallocatechin gallate (EGCG), penta-O-galloyl-&beta;-d-glucose (PGG), baicalein, oleuropein, and procyanidin B2. We find EGCG and PGG to be the best inhibitors. These compounds inhibit amyloid formation by redirecting the aggregation of FapC monomers into oligomeric species, which according to small-angle X-ray scattering (SAXS) measurements organize into core-shell complexes of short axis diameters 25&ndash;26 nm consisting of ~7 monomers. Using peptide arrays, we identify EGCG-binding sites in FapC&rsquo;s linker regions, C and N-terminal parts, and high amyloidogenic sequences located in the R2 and R3 repeats. We correlate our biophysical observations to biological impact by demonstrating that the extent of amyloid inhibition by the different inhibitors correlated with their ability to reduce biofilm, highlighting the potential of anti-amyloid polyphenols as therapeutic agents against biofilm infections

DIBMA nanodiscs keep α-synuclein folded

α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.info:eu-repo/semantics/publishedVersio

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization

[Image: see text] α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation

Self-assembling properties of ionisable amphiphilic drugs in aqueous solution

Hypothesis: Common amphiphilic drug molecules often have a more rigid nonpolar part than conventional surfactants. The rigidity is expected to influence the self-assembling properties and possibly give rise to aggregation patterns different from that of regular surfactants. Experiments: We have investigated self-assembling properties of the hydrochloride salts of adiphenine (ADP), pavatrine (PVT), and amitriptyline (AMT) at concentrations up to 50 wt% using small-angle x-ray scattering, dynamic light scattering, cryo-transmission electron microscopy, and surface tension measurements. Findings: All drugs form small micelles of oblate spheroidal shape at concentrations above the critical micelle concentrations (CMC). The micelles grow weakly in size up to about 20 wt%, where the aggregation number reaches a maximum followed by a slight decrease in size at higher drug concentrations. We observe a correlation between the decrease in micelle size at high concentrations and an increasing charge of the micelles, as the degree of ionization increases with increasing drug concentration and decreasing pH. In contrast to what has previously been reported, the aggregation behavior of all studied drugs resembles the closed association behavior of conventional surfactants with a short aliphatic chain as hydrophobic tail group i.e. the micelles are always small in size and lack a second CMC. CMC values were determined with surface tension measurements, including also lidocaine hydrochloride (LDC) and chlorpromazine hydrochloride (CHL)

Universal effective interactions of globular proteins close to liquid-liquid phase separation: corresponding-states behavior reflected in the structure factor

Intermolecular interactions in protein solutions in general contain many contributions. If short-range attractions dominate, the state diagram exhibits liquid-liquid phase separation (LLPS) that is metastable with respect to crystallization. In this case, the extended law of corresponding states (ELCS) suggests that thermodynamic properties are insensitive to details of the underlying interaction potential. Using lysozyme solutions, we investigate the applicability of the ELCS to the static structure factor and in how far effective colloidal interaction models can help to rationalize the phase behavior and interactions of protein solutions in the vicinity of the LLPS binodal. The (effective) structure factor has been determined by small-angle X-ray scattering (SAXS). It can be described by Baxter's adhesive hard-sphere model, which implies a single fit parameter from which the normalized second virial coefficient $b_2$ is inferred and found to quantitatively agree with previous results from static light scattering. The $b_2$ values are independent of protein concentration, but systematically vary with temperature and solution composition, i.e. salt and additive content. If plotted as a function of temperature normalized by the critical temperature, the values of $b_2$ follow a universal behaviour. These findings validate the applicability of the ELCS to globular protein solutions and indicate that the ELCS can also be reflected in the structure factor.Comment: The following article has been accepted by The Journal of Chemical Physics. After it is published, it will be found at https://doi.org/10.1063/5.008860

Bi-directional protein-protein interactions control liquid-liquid phase separation of PSD-95 and its interaction partners

The organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein-protein interactions (PPIs) which constitute a functional post-synapse. Postsynaptic density protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMAPR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the stargazin (TARP-γ2) mediated liquid-liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions while in other cases short peptides carrying a high charge are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems