130 results found
Ying L, Tahirbegi IB, Magness A, et al., 2022, Towards high throughput oligomer detection and classification for early-stage aggregation of amyloidogenic protein, Frontiers in Chemistry, ISSN: 2296-2646
The aggregation kinetics of proteins and peptides have been studied extensively due to theirsignificance in many human diseases, including neurodegenerative disorders, and the rolesthey play in some key physiological processes. However, most of these studies have beenperformed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents asindicators of fibrillization. Such techniques are highly successful in making inferences aboutthe nucleation and growth mechanism of fibrils yet cannot directly measure assembly reactionsat low protein concentrations which is the case for amyloid-β (Aβ) peptide under physiologicalconditions. In particular, the evolution from monomer to low order oligomer in the early stagesof aggregation cannot be detected. Single molecule methods allow directly access to suchfundamental information. We developed a high throughput protocol for single moleculephotobleaching experiments using an automated fluorescence microscope. Stepwisephotobleaching analysis of the time profiles of individual foci allowed us to determine thestoichiometry of the protein oligomers and probe protein aggregation kinetics. Furthermore,we investigated the potential application of supervised machine learning with support vectormachines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classifybleaching traces into stoichiometric categories based on an ensemble of measurablequantities derivable from individual traces. Both SVM and MLP models achieved comparableaccuracy of more than 80% against simulated traces up to 19-mer, although MLP offeredconsiderable speed advantages thus making it suitable for application to high throughputexperimental data. We used our high throughput method to study the aggregation of Aβ40 inthe presence of metal ions and the aggregation of α-synuclein in the presence of goldnanoparticles.
Ying L, Teng X, Sheveleva A, et al., 2021, Acetylation rather than H50Q mutation impacts the kinetics of Cu(II) binding to α-synuclein, ChemPhysChem: a European journal of chemical physics and physical chemistry, Vol: 22, Pages: 2413-2419, ISSN: 1439-4235
The interaction between α-synuclein (αSyn) and Cu2+ has been suggested to be closely linked to brain copper homeostasis. Disruption of copper levels could induce misfolding and aggregation of αSyn, and thus contribute to the progression of Parkinson's disease (PD). Understanding the molecular mechanism of αSyn-Cu2+ interaction is important and controversies in Cu2+ coordination geometry with αSyn still exists. Herein, we find that the pathological H50Q mutation has no impact on the kinetics of Cu2+ binding to the high-affinity site of wild type αSyn (WT-αSyn), indicating the non-involvement of His50 in high-affinity Cu2+ binding to WT-αSyn. In contrast, the physiological N-terminally acetylated αSyn (NAc-αSyn) displays several orders of magnitude weaker Cu2+ binding affinity than WT-αSyn. Cu2+ coordination mode to NAc-αSyn has also been proposed based on EPR spectrum. In addition, we find that Cu2+ coordinated WT-αSyn is reduction-active in the presence of GSH, but essentially inactive towards ascorbate. Our work provides new insights into αSyn-Cu2+ interaction, which may help understand the multifaceted normal functions of αSyn as well as pathological consequences of αSyn aggregation.
Teng X, Sheveleva A, Tuna F, et al., 2021, Front Cover: Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to α‐Synuclein (ChemPhysChem 23/2021), ChemPhysChem, Vol: 22, Pages: 2378-2378, ISSN: 1439-4235
Teng X, Sheveleva A, Tuna F, et al., 2021, Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to alpha-Synuclein, CHEMPHYSCHEM, ISSN: 1439-4235
Vilar R, Priessner M, Summers PA, et al., 2021, Selective detection of Cu+ ions in live cells via fluorescence lifetime imaging microscopy., Angewandte Chemie International Edition, Vol: 60, Pages: 23148-23153, ISSN: 1433-7851
Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).
Teng X, 2021, Probing the Interactions of Intrinsically Disordered Protein with Metal Ions and Lipid Membranes by Fluorescence Spectroscopy, 65th Annual Meeting of The Biophysical Society
Teng X, Willison K, Ying L, 2021, Probing the Interactions of Intrinsically Disordered Protein with Metal Ions and Lipid Membranes by Fluorescence Spectroscopy, 65th Annual Meeting of The Biophysical Society
Man W, Tahirbegi B, Vrettas M, et al., 2021, The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition, Nature Communications, Vol: 12, ISSN: 2041-1723
α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson’s disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.
Piersimoni ME, Teng X, Cass AEG, et al., 2020, Antioxidant lipoic acid ligand-shell gold nanoconjugates against oxidative stress caused by α-Synuclein aggregates, Nanoscale Advances, Vol: 2, Pages: 5666-5681, ISSN: 2516-0230
Gold nanoparticle is becoming a promising platform for the delivery of drugs to treat neurodegenerative diseases. Parkinson’s disease, associated with the aggregation of α-synuclein, is a condition that results in dysfunctional neuronal cells leading to their degeneration and death. Oxidative stress has been strongly implicated as a common feature in this process. The limited efficacy of the traditional therapies and the development of associated severe side effects present an unmet need for preventive and adjuvant therapies. The organosulfur compound lipoic acid, naturally located in the mitochondria, plays a powerful antioxidative role against oxidative stress. However, the efficacy is limited by its low physiological concentration, and the administration is affected by its short half-life and bioavailability due to hepatic degradation. Here we exploited the drug delivery potential of gold nanoparticles to assemble lipoic acid, and administered the system to SH-SY5Y cells, a cellular model commonly used to study Parkinson’s disease. We tested the nanoconjugates, termed GNPs-LA, under an oxidative environment induced by gold nanoparticle/α-synuclein conjugates (GNPs-α-Syn). GNPs-LA were found to be biocompatible and capable of restoring the cell damage caused by high-level reactive oxygen species generated by excessive oxidative stress in the cellular environment. We conclude that GNPs-LA may serve as a promising drug delivery vehicle conveying antioxidant molecules for the treatment of Parkinson’s disease.
Ying L, Tahirbegi B, Magness A, et al., 2020, A novel Aβ40 assembly at physiological concentration, Scientific Reports, Vol: 10, ISSN: 2045-2322
Aggregates of amyloid-β (Aβ) are characteristic of Alzheimer’s disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aβ. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aβ to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.
Le TT, Bruckbauer A, Tahirbegi B, et al., 2020, A highly stable RNA aptamer probe for the retinoblastoma protein in live cells, Chemical Science, Vol: 11, Pages: 4467-4474, ISSN: 2041-6520
<p>An RNA G-quadruplex aptamer, specific for the human retinoblastoma protein (RB) and highly stable inside cells, is selected and its application to live cell probing of the protein illustrated.</p>
Teng X, Stefaniak E, Girvan P, et al., 2020, Hierarchical binding of copperII to N-truncated Aβ4-16 peptide, Metallomics: integrated biometal science, Vol: 12, Pages: 470-473, ISSN: 1756-5901
N-Truncated Aβ4–42 displays a high binding affinity with CuII. A mechanistic scheme of the interactions between Aβ4–42 and CuII has been proposed using a fluorescence approach. The timescales of different conversion steps were determined. This kinetic mechanism indicates the potential synaptic functions of Aβ4–42 during neurotransmission.
Li X, Cui J, Wu L, et al., 2019, Bv8 contributes to neutrophil infiltration and triggers the angiogenesis of colon cancer via extracellular signal-regulated kinase-vascular endothelial growth factor signalling pathway, BJA Research Forum, Publisher: ELSEVIER SCI LTD, Pages: E500-E500, ISSN: 0007-0912
Gilburt J, Girvan P, Blagg J, et al., 2019, Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation, Chemical Science, Vol: 10, Pages: 4069-4076, ISSN: 2041-6520
Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes – such as protein kinases – adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand Kd, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.
Girvan P, Teng X, Brooks NJ, et al., 2019, Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications, 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
Zhou W, Hu L, Ying L, et al., 2018, A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection, Nature Communications, Vol: 9, ISSN: 2041-1723
Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.
Girvan P, Teng X, Brooks N, et al., 2018, Redox kinetics of the amyloid-β-Cu complex and its biological implications, Biochemistry, Vol: 57, Pages: 6228-6233, ISSN: 1520-4995
The ability of the amyloid-β peptide to bind to redox active metals and act as a source of radical damage in Alzheimer’s disease has been largely accepted as contributing to the disease’s pathogenesis. However, a kinetic understanding of the molecular mechanism, which underpins this radical generation, has yet to be reported. Here we use a sensitive fluorescence approach, which reports on the oxidation state of the metal bound to the amyloid-β peptide and can therefore shed light on the redox kinetics. We confirm that the redox goes via a low populated, reactive intermediate and that the reaction proceeds via the Component I coordination environment rather than Component II. We also show that while the reduction step readily occurs (on the 10 ms time scale) it is the oxidation step that is rate-limiting for redox cycling.
Zhou W, Cui H, Ying L, et al., 2018, Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing, Angewandte Chemie, Vol: 130, Pages: 10425-10429, ISSN: 0044-8249
Zhou W, Cui H, Ying L, et al., 2018, Enhanced cytosolic delivery and release of CRISPR/Cas9 by black phosphorus nanosheets for genome editing, Angewandte Chemie International Edition, Vol: 57, Pages: 10268-10272, ISSN: 1521-3757
A biodegradable two-dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered with three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3-BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation-associated endosomal escape and cytosolic releases of the loaded Cas9N3 complexes. The Cas9N3-BPs thus provide efficient genome editing and gene silencing in vitro and in vivo at a relatively low dose as compared with other nanoparticle-based delivery platforms. This biodegradable 2D delivery platform offers a versatile cytosolic delivery approach for CRISPR/Cas9 ribonucleoprotein and other bioactive macromolecules for biomedical applications.
Tahirbegi B, Magness AJ, Boillat A, et al., 2018, Probing synaptic amyloid-beta aggregation promoted by copper release, 62nd Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 430A-430A, ISSN: 0006-3495
Whether or not the metal ions released during synaptic transmission induce amyloid-beta oligomer formation in the vicinity of synapses is a central question pertinent to the molecular mechanism of Alzheimer's disease. Recently, through a combination of experimental kinetics studies and coupled reaction-diffusion simulations, we predicted that Cu(II) rather than Zn(II) plays an important role in the very early stages (i.e., dimer formation) of Aβ aggregation in the synapse. Single molecule photobleaching analysis is a powerful tool to determine the stoichiometry of amyloid-beta oligomers which enables us to examine the time course of small amyloid-beta oligomer formation in solution, immobilised to a solid-phase substrate or artificial lipid membrane, and in live neurons in the presence of Cu(II). Preliminary results indicate that small amyloid-beta oligomers can be locked in their oligomeric state without dissociation on a poly-lysine coated surface and that Cu(II) increases the diversity and abundance of amyloid-beta oligomers.
Fusco G, Chen SW, Williamson PTF, et al., 2017, Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers, Science, Vol: 358, Pages: 1440-1443, ISSN: 0036-8075
Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.
Gilburt JAH, Sarkar H, Sheldrake P, et al., 2017, Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy, Angewandte Chemie, Vol: 129, Pages: 11567-11572, ISSN: 0044-8249
The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure-based drug discovery.
Gilburt JAH, Sarkar H, Sheldrake P, et al., 2017, Dynamic equilibrium of Aurora-A kinase activation loop revealed by single molecule spectroscopy, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S315-S315, ISSN: 0175-7571
Branch T, Barahona M, Dodson C, et al., 2017, Kinetic analysis reveals the identity of Aβ-metal complex responsible for the initial aggregation of Aβ in the synapse, ACS Chemical Neuroscience, Vol: 8, Pages: 1970-1979, ISSN: 1948-7193
The mechanism of Aβ aggregation in the absence of metal ions is well established, yet the role that Zn2+ and Cu2+, the two most studied metal ions, released during neurotransmission, paly in promoting Aβ aggregation in the vicinity of neuronal synapses remains elusive. Here we report the kinetics of Zn2+ binding to Aβ and Zn2+/Cu2+ binding to Aβ-Cu to form ternary complexes under near physiological conditions (nM Aβ, μM metal ions). We find that these reactions are several orders of magnitude slower than Cu2+ binding to Aβ. Coupled reaction-diffusion simulations of the interactions of synaptically released metal ions with Aβ show that up to a third of Aβ is Cu2+-bound under repetitive metal ion release, while any other Aβ-metal complexes (including Aβ-Zn) are insignificant. We therefore conclude that Zn2+ is unlikely to play an important role in the very early stages (i.e., dimer formation) of Aβ aggregation, contrary to a widely held view in the subject. We propose that targeting the specific interactions between Cu2+ and Aβ may be a viable option in drug development efforts for early stages of AD.
Vilar Compte R, Klejevskaja B, Pyne LBA, et al., 2016, Studies of G-quadruplexes formed within self-assembled DNA mini-circles, Chemical Communications (London), Vol: 52, Pages: 12454-12457, ISSN: 0009-241X
We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.
Saleh AF, Fellows MM, Ying L, et al., 2016, The lack of mutagenic potential of a guanine-rich triplex forming oligonucleotide in physiological conditions, Toxicological Sciences, Vol: 155, Pages: 101-111, ISSN: 1096-6080
Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analysed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.
Girvan P, Miyake T, Teng X, et al., 2016, Kinetics of the Interactions between Copper and Amyloid-β with FAD Mutations and Phosphorylation at the N-terminus, Chembiochem, Vol: 17, Pages: 1732-1737, ISSN: 1439-7633
Mutations and post-translational modifications of amyloid-β (Aβ) peptide in its N terminus have been shown to increase fibril formation, yet the molecular mechanism is not clear. Here we investigated the kinetics of the interactions of copper with two Aβ peptides containing Familial Alzheimer's disease (FAD) mutations (English (H6R) and Tottori (D7N)), as well as with Aβ peptide phosphorylated at serine 8 (pS8). All three peptides bind to copper with a similar rate as the wild-type (wt). The dissociation rates follow the order pS8>H6R>wt>D7N; the interconversion between the two coordinating species occurs 50 % faster for H6R and pS8, whereas D7N had only a negligible effect. Interestingly, the rate of ternary complex (copper-bridged heterodimer) formation for the modified peptides was significantly faster than that for wt, thus leading us to propose that FAD and sporadic AD might share a kinetic origin for the enhanced oligomerisation of Aβ.
McFarlane C, Girvan P, Branch T, et al., 2016, Efficient lipid peroxidation catalyzed by amyloid-beta-copper complex: observation of chemical oscillation and chaos, 60th Annual Meeting of the Biophysical Society, Publisher: Biophysical Society, Pages: 552A-552A, ISSN: 1542-0086
Branch T, Girvan P, Barahona M, et al., 2015, Kinetics of amyloid-beta/metal ions interactions in the synaptic cleft: experiment and simulation, 10th EBSA European Biophysics Congress, Publisher: Springer Verlag, Pages: S230-S230, ISSN: 0175-7571
Mehta P, Jovanovic G, Ying L, et al., 2015, Is the cellular and molecular machinery docile in the stationary phase of Escherichia coli?, Biochemical Society Transactions, Vol: 43, Pages: 168-171, ISSN: 1470-8752
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