6 research outputs found
Role of Hydrophobicity and Charge of Amyloid-Beta Oligomer Eliminating dâPeptides in the Interaction with Amyloid-Beta Monomers
Inhibition of the
self-assembly process of amyloid-beta and even
more the removal of already existing toxic amyloid-beta assemblies
represent promising therapeutic strategies against Alzheimerâs
disease. To approach this aim, we selected a d-enantiomeric
peptide by phage-display based on the interaction with amyloid-beta
monomers. This lead compound was successfully optimized by peptide
microarrays with respect to its affinity and specificity to the target
resulting in d-peptides with both increased hydrophobicity
and net charge. Here, we present a detailed biophysical characterization
of the interactions between these optimized d-peptides and
amyloid-beta monomers in comparison to the original lead compound
in order to obtain a more thorough understanding of the physicochemical
determinants of the interactions. Kinetics and apparent stoichiometry
of complex formation were studied using surface plasmon resonance.
Potential modes of binding to amyloid-beta were identified, and the
influences of ionic strength on complex stability, as well as on the
inhibitory effect on amyloid-beta aggregation were investigated. The
results reveal a very different mode of interaction of the optimized d-peptides based on a combination of electrostatic and hydrophobic
interactions as compared to the mostly electrostatically driven interaction
of the lead compound. These conclusions were supported by the thermodynamic
profiles of the interaction between optimized d-peptides
and AÎČ monomers, which indicate an increase in binding entropy
with respect to the lead compound
Detection and Characterization of Small Molecule Interactions with Fibrillar Protein Aggregates Using Microscale Thermophoresis
Neurodegenerative
diseases such as Parkinsonâs and Alzheimerâs disease
share the pathological hallmark of fibrillar protein aggregates. The
specific detection of these protein aggregates by positron emission
tomography (PET) in the patient brain can yield valuable information
for diagnosis and disease progression. However, the identification
of novel small compounds that bind fibrillar protein aggregates has
been a challenge. In this study, microscale thermophoresis (MST) was
applied to assess the binding affinity of known small molecule ligands
of α-synuclein fibrils, which were also tested in parallel in
a thioflavin T fluorescence competition assay for further validation.
In addition, a MST assay was also developed for the detection of the
interaction between a variety of small molecules and tau fibrils.
The results of this study demonstrate that MST is a powerful and practical
methodology to quantify interactions between small molecules and protein
fibrillar aggregates, which suggests that it can be applied for the
identification and development of PET radioligands and potentially
of therapeutic candidates for protein misfolding diseases
Expanding the Solvent Chemical Space for Self-Assembly of Dipeptide Nanostructures
Nanostructures composed of short, noncyclic peptides represent a growing field of research in nanotechnology due to their ease of production, often remarkable material properties, and biocompatibility. Such structures have so far been almost exclusively obtained through self-assembly from aqueous solution, and their morphologies are determined by the interactions between building blocks as well as interactions between building blocks and water. Using the diphenylalanine system, we demonstrate here that, in order to achieve structural and morphological control, a change in the solvent environment represents a simple and convenient alternative strategy to the chemical modification of the building blocks. Diphenylalanine (FF) is a dipeptide capable of self-assembly in aqueous solution into needle-like hollow micro- and nanocrystals with continuous nanoscale channels that possess advantageous properties such as high stiffness and piezoelectricity and have so emerged as attractive candidates for functional nanomaterials. We investigate systematically the solubility of diphenylalanine in a range of organic solvents and probe the role of the solvent in the kinetics of self-assembly and the structures of the final materials. Finally, we report the crystal structure of the FF peptide in microcrystalline form grown from MeOH solution at 1 Ă
resolution and discuss the structural changes relative to the conventional materials self-assembled in aqueous solution. These findings provide a significant expansion of the structures and morphologies that are accessible through FF self-assembly for existing and future nanotechnological applications of this peptide. Solvent mediation of molecular recognition and self-association processes represents an important route to the design of new supramolecular architectures deriving their functionality from the nanoscale ordering of their components
Thermodynamics of Polypeptide Supramolecular Assembly in the Short-Chain Limit
The
self-assembly of peptides into ordered supramolecular structures,
such as fibrils and crystals, is of relevance in such diverse areas
as molecular medicine and materials science. However, little information
is available about the fundamental thermodynamic driving forces of
these types of self-assembly processes. Here, we investigate in detail
the thermodynamics of assembly of diphenylalanine (FF). This dipeptide
forms the central motif of the AÎČ peptides, which are associated
with Alzheimerâs disease through their presence in amyloid
plaques in the nervous systems of affected individuals. We identify
the molecular origins of the self-assembly of FF in aqueous solution,
and we evaluate these findings in the context of the aggregation free
energies of longer peptides that are able to form amyloid fibrils.
We find that the thermodynamics of FF assembly displays the typical
characteristics of hydrophobic desolvation processes, and detailed
analysis of the temperature dependence of the kinetics of assembly
within the framework of crystallization theories reveals that the
transition state from solution to crystalline aggregates is enthalpically
unfavorable and entropically favorable, qualitatively similar to what
has been found for longer sequences. This quantitative comparison
of aggregating peptides of very different lengths is the basis of
an in-depth understanding of the relationship between sequence and
assembly behavior
Microfluidic Diffusion Platform for Characterizing the Sizes of Lipid Vesicles and the Thermodynamics of ProteinâLipid Interactions
Elucidation of the
fundamental interactions of proteins with biological
membranes under native conditions is crucial for understanding the
molecular basis of their biological function and malfunction. Notably,
the large surface to volume ratio of living cells provides a molecular
landscape for significant interactions of cellular components with
membranes, thereby potentially modulating their function. However,
such interactions can be challenging to probe using conventional biophysical
methods due to the heterogeneity of the species and processes involved.
Here, we use direct measurements of micron scale molecular diffusivity
to detect and quantify the interactions of α-synuclein, associated
with the etiology of Parkinsonâs disease, with negatively charged
lipid vesicles. We further demonstrate that this microfluidic approach
enables the characterization of size distributions of different binary
mixtures of vesicles, which are not readily accessible using conventional
light scattering techniques. Finally, the size distributions of the
two α-synuclein conformations, free α-synuclein and membrane-bound
α-synuclein, were resolved under varying lipid:protein ratios,
thus, allowing the determination of the dissociation constant and
the binding stoichiometry associated with this proteinâlipid
system. The microfluidic diffusional sizing platform allows these
measurements to be performed on a time scale of minutes using microlitre
volumes, thus, establishing the basis for an approach for the study
of molecular interactions of heterogeneous systems under native conditions
ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.
<p><b>A.</b> ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 ”g/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087133#pone.0087133.s001" target="_blank">file S1</a>). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 ÎŒ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (nâ=â3+/â s.e.m *pâ€0.001 versus no compound Tet-induced sample). <b>B.</b> Microglia isolated from postnatal day 1 to 3 pups from hSNCA<sup>E46K</sup> transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 ”M ELN484217 or ELN484228 followed by addition of 10 ”m beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (nâ=â3+/â s.e.m *pâ€0.001). <b>C.</b> ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (âcontrolâ) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 ”M ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. nâ=â3 for neuron viability analysis; nâ=â160â206 for neurite length analysis. *p-valueâ€0.05, ***p-valueâ€0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. <b>D.</b> ELN484228 reduces translocation of αSyn to the phagocytic cup<b>.</b> To assess αSyn translocation, H4 cells were treated with 100 ”M ELN484228 and 1 ”g/ml tetracycline for 24 hours; cells were then stimulated with 4 ÎŒ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. <b>E.</b> ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (âŒ21DIV) grown in serum-free conditions were treated for 24 hours with 1 ”M ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 ”m. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/â SEM, nâ=â1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2â3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.</p