Deciphering the molecular signatures of neurodegeneration by predictive computational modelling

Amyloids are fibrillary aggregates identified in over 40 human diseases, including neurodegenerative diseases encompassing Alzheimer’s (AD), Parkinson’s (PD), Huntington’s and Prion diseases. Amyloids form by spontaneous self-assembly of monomeric precursor peptides known as intrinsically disordered proteins (IDPs). Experiments suggest that soluble low molecular weight oligomers formed in the early stages of assembly are toxic, and hence, most promising drug targets. However, experiments are insufficient to characterize oligomers due to their inherent polymorphic and short-lived nature. This thesis advances our mechanistic understanding of the formation of amyloid oligomers by delineating signature features of IDP monomers and ‘profibrillar’ oligomers through predictive computational modelling techniques employing atomic resolution molecular dynamics (MD) computer simulations. We additionally predict the assembly of non-aggregating low molecular weight oligomers. We first probe the molecular signatures of experimentally indicative non-aggregating folded a-helical conformers, and aggregation-prone partially folded a-helices of amyloid-b42 (Ab42) and a-synuclein (aS) IDPs implicated in AD and PD, respectively across a broad spectrum of physical models. We predict a common intra-peptide route to helix stabilization, showing that the terminal groups (N-terminal or NTR in Ab42 and C terminal or CTR in aS) frequently indulge in hydrophobic interactions with the central hydrophobic domains (CHDs) and secondary salt bridges with other domains. Lack of such short-range contacts during complete helix unfolding coupled with destabilized helices in terminal-deleted variants confer the aggregation protective role by terminal groups in folded helical conformers. Further, we reveal a shared feature of dynamic coupling between the partially folded helical regions of the CHD and the charged terminal ends (NTR in Ab42 and CTR in aS). Absence of such intra-peptide modulation in helically folded and unfolded states confer long-range allosteric regulation of the CHD by the termini that may render the partially folded helical states prone to initial oligomerization. Next, we design structural assemblies of experimentally uncharacterized aggregation-resistant low-weight aS tetramer. We model a de novo broken a-helical tetramer by reconstructing loop motif that optimizes packing of aS helical monomers. We show that monomers attain activated conformations during tetramer assembly, and familial missense mutations double the energy barrier to tetramerization, thus preserving the pool of aggregation-prone disordered monomers, and confirming the experimentally observed low tetramer:monomer ratios with mutants. In order to investigate the effect of helical continuity and periodicity, we model a de novo extended 11/3-helical tetramer. Broken a-helical tetramers show a more favourable assembly than the extended 11/3- helical tetramers, the ease of their interconversions diminishing with homologous E → K mutations. Additionally, rationally designing a series of broken a-helical multimers from dimers to octamers shows that tetramers have lowest activation energy, providing a rationale for the experimental observation that tetramers are the most populated oligomers. Finally, we investigate the molecular determinants of higher aggregation rate of Ab42 over Ab40 by simulating their profibrillar oligomers (dodecamers) on graphene water interface. Our data reveals that Ab dodecamers may facilitate a single layer growth along the graphene surface, with Ab42 presenting a more closed conformation with possibilities of unidirectional growth in Ab40, but not in Ab42. Oligomer height profiles on graphene indicate that dodecamers may be formed post mature fibril formatio

On the ubiquity of helical α-synuclein tetramers

The experimental finding that α-synuclein (αS) occurs physiologically as a helically folded tetramer begs the question: why are helical tetramers the most populated multimers? While the helical tetramer is known to resist aggregation, the assembly mechanism of αS peptides remains largely unknown. By rationally designing a series of helical multimers from dimer to octamer, we characterized the free energy landscape of wild-type and mutated multimers using molecular dynamics computer simulations. Competition between supramolecular packing and solvation results in well-hydrated dimers and trimers, and more screened pentamers to octamers, with the helical tetramer possessing the most balanced structure with the lowest activation energy. Our data suggest that familial mutants are very sensitive to alterations in monomer packing that would in turn raise the energy barriers for multimerization. Finally, the hypothesis that the αS tetramer forms a soluble, benign “dead end” to circumvent aggregation is supported by its computed very weak association with negatively charged cell membranes

Rational design of therapeutic peptides using physics-based molecular dynamics simulations

Abstract: Peptides are sustainable alternatives to conventional therapeutics for G protein-coupled receptor (GPCR) linked disorders, promising biocompatible and tailorable next-generation therapeutics for metabolic disorders including type-2 diabetes, as agonists of the glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R). However, single agonist peptides activating GLP-1R to stimulate insulin secretion also suppress obesity-linked glucagon release. Hence, bioactive peptides co-targeting GCGR and GLP-1R may remediate the blood glucose and fatty acid metabolism imbalance, tackling both diabetes and obesity to supersede current mono-agonist therapy. Here we design and model optimised peptide sequences starting from peptide sequences derived from earlier phage-displayed library screening, identifying those with predicted molecular binding profiles for dual agonism of GCGR and GLP-1R. We derive design rules from extensive molecular dynamics simulations based on peptide–receptor binding. Our newly designed co-agonist peptide exhibits improved coupled binding affinity for GCGR and GLP-1R relative to endogenous ligands, which may provide superior glycaemic and weight loss control.Event Details: https://www.ul.ie/node/85169UL PHYSICS DEPARTMENT RESEARCH DAY – 19th May 2023The event showcased the breadth of research conducted in the Department and was an excellent opportunity for networking and engaging with the Physics community at UL.</p

Coupled electrostatic and hydrophobic destabilisation of the gelsolin-actin complex enables facile detection of ovarian cancer biomarker lysophosphatidic acid

Lysophosphatidic acid (LPA) is a promising biomarker candidate to screen for ovarian cancer (OC) and potentially stratify and treat patients according to disease stage. LPA is known to target the actin-binding protein gelsolin which is a key regulator of actin filament assembly. Previous studies have shown that the phosphate headgroup of LPA alone is inadequate to bind to the short chain of amino acids in gelsolin known as the PIP2 -binding domain. Thus, the molecular-level detail of the mechanism of LPA binding is poorly understood. Here, we model LPA binding to the PIP2 - binding domain of gelsolin in the gelsolin-actin complex through extensive ten-microsecond atomistic molecular dynamics (MD) simulations. We predict that LPA binding causes a local conformational rearrangement due to LPA interactions with both gelsolin and actin residues. These conformational changes are a result of the amphipathic nature of LPA, where the anionic phosphate, polar glycerol and ester groups, and lipophilic aliphatic tail mediate LPA binding via charged electrostatic, hydrogen bonding, and van der Waals interactions. The negatively-charged LPA headgroup binds to the PIP2 - binding domain of gelsolin-actin while its hydrophobic tail is inserted into actin, creating a strong LPA-insertion pocket that weakens the gelsolin–actin interface. The computed structure, dynamics, and energetics of the ternary gelsolin–LPA–actin complex confirms that a quantitative OC assay is possible based on LPA-triggered actin release from the gelsolin-actin complex</p

Modelling peptide self-assembly within a partially disordered tau filament

Peptide self-assemblies are a natural template for designing bio-inspired functional materials given the extensive characterisation of neurodegenerative and non-disease biological amyloid protein assemblies and advances in rational, modelling-led materials design. These bioinspired materials employ design rules obtained from known aggregation-prone peptides or de novo screening for sequences most amenable to self-assemble functional nanostructures. Here, we exploit the hybrid nature of a complex peptide with both ordered crystalline and intrinsically disordered regions, namely, the microtubule-binding domain (MBD) of tau protein, to probe the physical driving forces for self-assembly at the molecular level. We model the peptide in its native and mutated states to identify the supramolecular packing driving stabilisation at the prefibrillar level. We use extensive atomic-resolution molecular dynamics computer simulations, contact maps, hydrogen-bond net-works and free energy calculations to model the tau MBD and its two known familial mutants, the P301L and K280Δ, along with a control double mutant, P301L + K280Δ as a first step towards understanding their effects on oligomer stability in fibrillar fold. Our results indicate that the mutations destabilise supramolecular packing in the pro-fibrillar hexamer by breaking contacts in the ordered domain of tau MBD, which helps explain mutation-induced toxicity levels as the more stable wild-type peptide assemblies may be less prone to crumbling, producing fewer toxic small oligomeric seeds. Our most important finding is that tau familial mutations causing frontotemporal dementia may show distinct morphologies delineating different stages of self-assembly. The models show that the P301L mutant is more pro-nucleating with low tendency for assembly polymerisation, whereas K280Δ is more pro-elongating with potential for protofibrillar growth. Our data provides a predictive mechanistic model for distinct peptide self-assembly features depending on the location and nature of single missense mutations on the partially disordered pathogenic MBD, which may explain the prevalence of polymorphic filamentous tau strains observed experimentally.</p

Label-free digital holotomography reveals ibuprofen-induced morphological changes to red blood cells

Understanding the dose-dependent effect of over-the-counter drugs on red blood cells (RBCs) is crucial for hematology and digital pathology. Yet, it is challenging to continuously record the real-time, drug-induced shape changes of RBCs in a label-free manner. Here, we demonstrate digital holotomography (DHTM)-enabled real-time, label-free concentration-dependent and time-dependent monitoring of ibuprofen on RBCs from a healthy donor. The RBCs are segmented based on three-dimensional (3D) and four-dimensional (4D) refractive index tomograms, and their morphological and chemical parameters are retrieved with their shapes classified using machine learning. We directly observed the formation and motion of spicules on the RBC membrane when aqueous solutions of ibuprofen were drop-cast on wet blood, creating rough-membraned echinocyte forms. At low concentrations of 0.25−0.50 mM, the ibuprofen-induced morphological change was transient, but at high concentrations (1−3 mM) the spiculated RBC remained over a period of up to 1.5 h. Molecular simulations confirmed that aggregates of ibuprofen molecules at high concentrations significantly disrupted the RBC membrane structural integrity and lipid order but produced negligible effect at low ibuprofen concentrations. Control experiments on the effect of urea, hydrogen peroxide, and aqueous solutions on RBCs showed zero spicule formation. Our work clarifies the dose-dependent chemical effects on RBCs using label-free microscopes that can be deployed for the rapid detection of overdosage of over-the-counter and prescribed drugs.</p

Conformational selection of α‑Synuclein tetramers at biological interfaces

Controlling the polymorphic assemblies of α-synuclein (αS) oligomers is crucial to reroute toxic protein aggregation implicated in Parkinson’s disease (PD). One potential mediator is the interaction of αS tetramers with cell membranes, which may regulate the dynamic balance between aggregation-prone disordered monomers and aggregation-resistant helical tetramers. Here, we model diverse tetramer−cell interactions and compare the structure−function relations at the supramolecular−biological interface with available experimental data. The models predict preferential interaction of compact αS tetramers with highly charged membrane surfaces, which may further stabilize this aggregation-resistant conformer. On moderately charged membranes, extended structures are preferred. In addition to surface charge, curvature influences tetramer thermodynamic stability and aggregation, with potential for selective isolation of tetramers via regio-specific interactions with strongly negatively charged micelles that screen further aggregation. Our modeling data set highlights diverse beneficial nano−bio interactions to redirect biomolecule assembly, supporting new therapeutic approaches for PD based on lipid-mediated conformational selection and inhibition</p

Solid lipid nanoparticle formulation maximizes membrane-damaging efficiency of antimicrobial nisin Z peptide

Solid lipid nanoparticles (SLNs) can protect and deliver naturally derived or synthetic biologically active products to target sites in vivo. Here, an SLN formulation produces a measured four-fold reduction in inhibitory concentration of an antimicrobial peptide nisin Z against S. aureus as compared to the free peptide, indicating the successful delivery and enhanced effectiveness of the SLN-encapsulated bacteriocin. Spherical SLNs of size 79.47 ± 2.01 nm and zeta potential of − 9.8 ± 0.3 mV were synthesised. The lipid formulation maximizes the membrane-damaging mode of action of the free peptide with more and larger-sized pores formed on bacterial membranes treated with nisin Z SLNs as measured from scanning electron microscopy and transmission electron microscopy. Flow cytometry measurements precisely quantified an enhanced dye leakage from pre-labeled bacterial cells when treated with nisin Z-loaded SLNs compared to free peptide. The lipid formulation accelerated cell death by killing all the cells within half an hour compared to the equivalent concentration of free peptide which was not bactericidal. Molecular dynamics simulations revealed a mechanism of SLN facilitated binding to the lipid II bacterial cell wall precursor via enhanced adsorption of nisin Z at the inner bacterial cell membrane bilayer. These findings confirmed the potential of SLN formulations for the effective delivery of therapeutic peptides for next-generation antibiotics that are active at low concentrations with the potential to mitigate antimicrobial resistance.</p

Tracking prenucleation molecular clustering of salicylamide in organic solvents

Crystal nucleation shapes the structure and product size distribution of solid-state pharmaceuticals and is seeded by early-stage molecular self-assemblies formed in host solution. Here, molecular clustering of salicylamide in ethyl acetate, methanol, and acetonitrile was investigated using photon correlation spectroscopy. Cluster size steadily increased over 3 days and with concentration across the range from undersaturated to supersaturated solutions. Solute concentration normalized by solubility provided more sensitive characterization of molecular-level conditions than concentration alone. In saturated solution, cluster size is independent of solvent, while at equal supersaturation, solvent-dependent cluster size increases as methanol < acetonitrile < ethyl acetate, commensurate with increasing nucleation propensity. In ethyl acetate, with largest prenucleation clusters, the driving force required for nucleation is lowest, compared to methanol with smallest clusters and highest driving force. To understand solvent− solute effects, we performed IR spectroscopy supported by molecular simulations. We observe solute−solvent interaction weakening in the same order: methanol < acetonitrile < ethyl acetate, quantifying the weaker solvent−solute interactions that permit the formation of larger prenucleation clusters. Our results support the hypothesis that nucleation is easier in weaker solvents because weak solute−solvent interactions favor growth of large clusters, as opposed to relying solely on ease of desolvation.</p

Single-Particle Resolution of Copper-Associated Annular α‑Synuclein Oligomers Reveals Potential Therapeutic Targets of Neurodegeneration

 Metal ions stabilize protein−protein interactions and can modulate protein aggregation. Here, using liquid-based atomic force microscopy and molecular dynamics simulations, we study the concentration-dependent effect of Cu2+ ions on the aggregation pathway of α-synuclein (α-Syn) proteins, which play a key role in the pathology of Parkinson’s disease. The full spectrum of α-Syn aggregates in the presence and absence of Cu2+ ions from monomers to mature fibrils was resolved and quantified at the gold−water interface. Raman spectroscopy confirmed the atomic force microscopy (AFM) findings on the heterogeneity in aggregated states of α-Syn. The formation of annular oligomers was exclusively detected upon incubating α-Syn with Cu2+ ions. Our findings emphasize the importance of targeting annular α-Syn protein oligomers for therapeutic intervention and their potential role as biomarkers for early detection and monitoring progression of neurodegeneration. </p