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

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

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

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

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

Computational peptide design cotargeting glucagon and glucagon-like peptide‑1 receptors

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 cotargeting GCGR and GLP-1R may remediate the blood glucose and fatty acid metabolism imbalance, tackling both diabetes and obesity to supersede current monoagonist therapy. Here, we design and model optimized 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 coagonist peptide exhibits improved predicted coupled binding affinity for GCGR and GLP-1R relative to endogenous ligands and could in the future be tested experimentally, which may provide superior glycemic and weight loss control.</p

Lithiophilic nanowire guided Li deposition in Li metal batteries

Lithium (Li) metal batteries (LMBs) provide superior energy densities far  beyond current Li-ion batteries (LIBs) but practical applications are hindered  by uncontrolled dendrite formation and the build-up of dead Li in “hostless”  Li metal anodes. To circumvent these issues, we created a 3D framework of  a carbon paper (CP) substrate decorated with lithiophilic nanowires (silicon  (Si), germanium (Ge), and SiGe alloy NWs) that provides a robust host for  efficient stripping/plating of Li metal. The lithiophilic Li22Si5, Li22(Si0.5Ge0.5)5, and Li22Ge5 formed during rapid Li melt infiltration prevented the forma?tion of dead Li and dendrites. Li22Ge5/Li covered CP hosts delivered the best  performance, with the lowest overpotentials of 40 mV (three times lower  than pristine Li) when cycled at 1 mA cm−2 /1 mAh cm−2  for 1000 h and at  3 mA cm−2 /3 mAh cm−2  for 500 h. Ex situ analysis confirmed the ability of  the lithiophilic Li22Ge5 decorated samples to facilitate uniform Li deposi?tion. When paired with sulfur, LiFePO4, and NMC811 cathodes, the CP-LiGe/ Li anodes delivered 200 cycles with 82%, 93%, and 90% capacity retention,  respectively. The discovery of the highly stable, lithiophilic NW decorated CP  hosts is a promising route toward stable cycling LMBs and provides a new  design motif for hosted Li metal anodes. </p

Hydroxychloroquine Does Not Function as a Direct Zinc Ionophore

Drug-mediated correction of abnormal biological zinc homeostasis could provide new routes to treating neurodegeneration, cancer, and viral infections. Designing therapeutics to facilitate zinc transport intracellularly is hampered by inadequate concentrations of endogenous zinc, which is often protein-bound in vivo. We found strong evidence that hydroxychloroquine, a drug used to treat malaria and employed as a potential treatment for COVID-19, does not bind and transport zinc across biological membranes through ionophoric mechanisms, contrary to recent claims. In vitro complexation studies and liposomal transport assays are correlated with cellular zinc assays in A549 lung epithelial cells to confirm the indirect mechanism of hydroxychloroquine-mediated elevation in intracellular zinc without ionophorism. Molecular simulations show hydroxychloroquine-triggered helix perturbation in zinc-finger protein without zinc chelation, a potential alternative non-ionophoric mechanism. </p

Biological effects of the loss of homochirality in a multicellular organism

Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism’s health, we use chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a ‘heterochirality syndrome’ with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.</p

Modulating the pro-apoptotic activity of cytochrome c at a biomimetic electrified interface

Programmed cell death via apoptosis is a natural defence against excessive cell division, crucial for fetal development to maintenance of homeostasis and elimination of precancerous and senescent cells. Here, we demonstrate an electrified liquid biointerface that replicates the molecular machinery of the inner mitochondrial membrane at the onset of apoptosis. By mimicking in vivo cytochrome c (Cyt c) interactions with cell membranes, our platform allows us to modulate the conformational plasticity of the protein by simply varying the electrochemical environment at an aqueous-organic interface. We observe interfacial electron transfer between an organic electron donor decamethylferrocene and O2, electrocatalyzed by Cyt c. This interfacial reaction requires partial Cyt c unfolding, mimicking Cyt c in vivo peroxidase activity. As proof of concept, we use our electrified liquid biointerface to identify drug molecules, such as bifonazole, that can potentially down-regulate Cyt c and protect against uncontrolled neuronal cell death in neurodegenerative disorders