114 results on '"Stephens, Amberley D."'
Search Results
2. Short hydrogen bonds enhance nonaromatic protein-related fluorescence
- Author
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Stephens, Amberley D., Qaisrani, Muhammad Nawaz, Ruggiero, Michael T., Mirón, Gonzalo Díaz, Morzan, Uriel N., Lebrero, Mariano C. González, Jones, Saul T. E., Poli, Emiliano, Bond, Andrew D., Woodhams, Philippa J., Kleist, Elyse M., Grisanti, Luca, Gebauer, Ralph, Zeitler, J. Axel, Credgington, Dan, Hassanali, Ali, and Schierle, Gabriele S. Kaminski
- Published
- 2021
3. Machine learning-based spike sorting reveals how subneuronal concentrations of monomeric Tau cause a loss in excitatory postsynaptic currents in hippocampal neurons
- Author
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Brockhoff, Marius, primary, Träuble, Jakob, additional, Middya, Sagnik, additional, Fuchsberger, Tanja, additional, Fernandez-Villegas, Ana, additional, Stephens, Amberley D, additional, Robbins, Miranda, additional, Dai, Wenyue, additional, Haider, Belquis, additional, Vora, Sulay, additional, Läubli, Nino F, additional, Kaminski, Clemens F, additional, Malliaras, George G, additional, Paulsen, Ole, additional, and Schierle, Gabriele S Kaminski, additional
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- 2024
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4. Observation of high-temperature macromolecular confinement in lyophilised protein formulations using terahertz spectroscopy
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Shmool, Talia A., Woodhams, P.J., Leutzsch, Markus, Stephens, Amberley D., Gaimann, Mario U., Mantle, Michael D., Kaminski Schierle, Gabriele S., van der Walle, Christopher F., and Zeitler, J. Axel
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- 2019
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5. Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein
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Seetaloo, Neeleema, Zacharopoulou, Maria, Stephens, Amberley D, Kaminski Schierle, Gabriele S, Phillips, Jonathan J, Seetaloo, Neeleema [0000-0002-8312-2542], Stephens, Amberley D [0000-0002-7303-6392], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], Phillips, Jonathan J [0000-0002-5361-9582], and Apollo - University of Cambridge Repository
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Protein Conformation ,alpha-Synuclein ,Humans ,Deuterium Exchange Measurement ,Hydrogen Deuterium Exchange-Mass Spectrometry ,Parkinson Disease ,Deuterium ,Article ,Analytical Chemistry - Abstract
In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.
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- 2022
6. Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
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Stephens, Amberley D., Zacharopoulou, Maria, Moons, Rani, Fusco, Giuliana, Seetaloo, Neeleema, Chiki, Anass, Woodhams, Philippa J., Mela, Ioanna, Lashuel, Hilal A., Phillips, Jonathan J., De Simone, Alfonso, Sobott, Frank, and Schierle, Gabriele S. Kaminski
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- 2020
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- View/download PDF
7. Decreased Water Mobility Contributes To Increased α‐Synuclein Aggregation**
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Stephens, Amberley D., primary, Kölbel, Johanna, additional, Moons, Rani, additional, Chung, Chyi Wei, additional, Ruggiero, Michael T., additional, Mahmoudi, Najet, additional, Shmool, Talia A., additional, McCoy, Thomas M., additional, Nietlispach, Daniel, additional, Routh, Alexander F., additional, Sobott, Frank, additional, Zeitler, J. Axel, additional, and Kaminski Schierle, Gabriele S., additional
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- 2023
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8. α-synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased uptake into neurons
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Stephens, Amberley D., primary, Villegas, Ana Fernandez, additional, Chung, Chyi Wei, additional, Vanderpoorten, Oliver, additional, Pinotsi, Dorothea, additional, Mela, Ioanna, additional, Ward, Edward, additional, McCoy, Thomas M., additional, Cubitt, Robert, additional, Routh, Alexander F., additional, Kaminski, Clemens F., additional, and Schierle, Gabriele S. Kaminski, additional
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- 2022
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9. Intracellular FUS protein accumulation leads to cytoskeletal, organelle and cellular homeostasis perturbations
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Chung, Chyi Wei, primary, Zhou, Alexandra J., additional, Mela, Ioanna, additional, Stephens, Amberley D., additional, Miyashita, Akinori, additional, St George-Hyslop, Peter H., additional, Kaminski, Clemens F., additional, Knowles, Tuomas P. J., additional, and Kaminski Schierle, Gabriele S., additional
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- 2022
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10. Intracellular Aβ42 Aggregation Leads to Cellular Thermogenesis
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Chung, Chyi Wei, primary, Stephens, Amberley D., additional, Konno, Tasuku, additional, Ward, Edward, additional, Avezov, Edward, additional, Kaminski, Clemens F., additional, Hassanali, Ali A., additional, and Kaminski Schierle, Gabriele S., additional
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- 2022
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11. Phylogeographic diversity and mosaicism of the Helicobacter pylori tfs integrative and conjugative elements
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Delahay, Robin M., Croxall, Nicola J., and Stephens, Amberley D.
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- 2018
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12. Correction: Corrigendum: Structural basis of synaptic vesicle assembly promoted by α-synuclein
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Fusco, Giuliana, Pape, Tillmann, Stephens, Amberley D., Mahou, Pierre, Costa, Ana Rita, Kaminski, Clemens F., Schierle, Gabriele S. Kaminski, Vendruscolo, Michele, Veglia, Gianluigi, Dobson, Christopher M., and De Simone, Alfonso
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- 2017
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13. Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property
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Chung, Chyi Wei, primary, Stephens, Amberley D., additional, Ward, Edward, additional, Feng, Yuqing, additional, Davis, Molly Jo, additional, Kaminski, Clemens F., additional, and Kaminski Schierle, Gabriele S., additional
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- 2022
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14. Local structural dynamics of alpha-synuclein correlate with aggregation in different physiological conditions
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Seetaloo, Neeleema, primary, Zacharopoulou, Maria, additional, Stephens, Amberley D., additional, Kaminski Schierle, Gabriele S., additional, and Phillips, Jonathan J., additional
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- 2022
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15. Label-free characterisation of amyloids and alpha-Synuclein polymorphs by exploiting their intrinsic fluorescence property
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Chung, Chyi Wei, primary, Stephens, Amberley D, additional, Ward, Edward, additional, Feng, Yuqing, additional, Davis, Molly Jo, additional, Kaminski, Clemens F, additional, and Kaminski Schierle, Gabriele S, additional
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- 2021
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- View/download PDF
16. C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction
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Lautenschläger, Janin, Stephens, Amberley D., Fusco, Giuliana, Ströhl, Florian, Curry, Nathan, Zacharopoulou, Maria, Michel, Claire H., Laine, Romain, Nespovitaya, Nadezhda, Fantham, Marcus, Pinotsi, Dorothea, Zago, Wagner, Fraser, Paul, Tandon, Anurag, St George-Hyslop, Peter, Rees, Eric, Phillips, Jonathan J., De Simone, Alfonso, Kaminski, Clemens F., Kaminski Schierle, Gabriele S., Stephens, Amberley D [0000-0002-7303-6392], Ströhl, Florian [0000-0002-2603-0780], St George-Hyslop, Peter [0000-0003-0796-7209], Phillips, Jonathan J [0000-0002-5361-9582], Kaminski, Clemens F [0000-0002-5194-0962], Schierle, Gabriele S Kaminski [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
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animal diseases ,Science ,Presynaptic Terminals ,In Vitro Techniques ,Article ,Cell Line ,Rats, Sprague-Dawley ,Protein Aggregates ,Microscopy, Electron, Transmission ,mental disorders ,Animals ,Humans ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,Binding Sites ,Lipid Metabolism ,Rats ,nervous system diseases ,nervous system ,alpha-Synuclein ,lcsh:Q ,Calcium ,Synaptic Vesicles ,Protein Binding ,Synaptosomes - Abstract
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies., Alpha-synuclein is associated with neuronal dysfunction in Parkinson’s disease. This study shows that alpha-synuclein interacts with neuronal synaptic vesicles in a calcium-dependent fashion, and this interaction is important for synaptic vesicle clustering.
- Published
- 2018
17. The role of water mobility in protein misfolding
- Author
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Stephens, Amberley D., primary, Kölbel, Johanna, additional, Moons, Rani, additional, Ruggerio, Michael T., additional, Mahmoudi, Najet, additional, Shmool, Talia A., additional, McCoy, Thomas M., additional, Nietlispach, Daniel, additional, Routh, Alexander F., additional, Sobott, Frank, additional, Zeitler, J. Axel, additional, and Schierle, Gabriele S. Kaminski, additional
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- 2021
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18. Purification of Recombinant α-synuclein: A Comparison of Commonly Used Protocols
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Stephens, Amberley D., primary, Matak-Vinkovic, Dijana, additional, Fernandez-Villegas, Ana, additional, and Kaminski Schierle, Gabriele S., additional
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- 2020
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19. Intrinsic fluorescence in non-aromatic peptide structures is induced by collective vibrations, charge reorganisation and short hydrogen bonds, as shown in a new glutamine-related structure
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Stephens, Amberley D., Qaisrani, Muhammad Nawaz, Ruggiero, Michael T., Jones, Saul T.E., Poli, Emiliano, Bond, Andrew D., Woodhams, Philippa J., Kleist, Elyse M., Grisanti, Luca, Gebauer, Ralph, Zeitler, J. Axel, Credgington, Dan, Hassanali, Ali, and Kaminski Schierle, Gabriele S.
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fluorescence, hydrogen bonds, peptide - Abstract
Disentangling the origin of the optical activity of non-aromatic proteins is challenging due to their size and thus their high computational requisites. Here we show, in a much smaller model system, that the single amino acid glutamine undergoes a chemical transformation leading to an unreported glutamine-like structure which has a similar broad absorption spectrum reported previously for non-aromatic proteins. We further show computationally that the optical activity of the glutamine-like structure is directly coupled to short-hydrogen bonds, but also displays charge and vibrational fluctuations, the latter of which are also present in less optically active structures such as in L-glutamine. Since experimentally the glutamine-like structure is the brightest structure, we conclude that short-hydrogen bonds are the ones responsible for the large Stokes shift observed in optically active non-aromatic proteins.
- Published
- 2020
20. Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing
- Author
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Stephens, Amberley D., Nespovitaya, Nadezhda, Zacharopoulou, Maria, Kaminski, Clemens F., Phillips, Jonathan J., and Kaminski Schierle, Gabriele S.
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Protein Conformation ,Freezing ,alpha-Synuclein ,Humans ,Parkinson Disease ,Article - Abstract
Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson's and Alzheimer's disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.
- Published
- 2018
21. Fast Purification of Recombinant Monomeric Amyloid-β from E. coli and Amyloid-β-mCherry Aggregates from Mammalian Cells
- Author
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Stephens, Amberley D., primary, Lu, Meng, additional, Fernandez-Villegas, Ana, additional, and Kaminski Schierle, Gabriele S., additional
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- 2020
- Full Text
- View/download PDF
22. Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies
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Lautenschläger, Janin, primary, Wagner-Valladolid, Sara, additional, Stephens, Amberley D., additional, Fernández-Villegas, Ana, additional, Hockings, Colin, additional, Mishra, Ajay, additional, Manton, James D., additional, Fantham, Marcus J., additional, Lu, Meng, additional, Rees, Eric J., additional, Kaminski, Clemens F., additional, and Kaminski Schierle, Gabriele S., additional
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- 2020
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- View/download PDF
23. Fast purification of recombinant monomeric amyloid-β from E. coli and amyloid-β-mCherry aggregates from mammalian cells
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Stephens, Amberley D, primary, Lu, Meng, additional, and Kaminski Schierle, Gabriele S, additional
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- 2020
- Full Text
- View/download PDF
24. Short hydrogen bonds enhance non-aromatic protein-related fluorescence
- Author
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Stephens, Amberley D., primary, Qaisrani, Muhammad Nawaz, additional, Ruggiero, Michael T., additional, Miron, Gonzalo Diaz, additional, Morzan, Uriel N., additional, González Lebrero, Mariano C., additional, Jones, Saul T.E., additional, Poli, Emiliano, additional, Bond, Andrew D., additional, Woodhams, Philippa J., additional, Kleist, Elyse M., additional, Grisanti, Luca, additional, Gebauer, Ralph, additional, Zeitler, J. Axel, additional, Credgington, Dan, additional, Hassanali, Ali, additional, and Schierle, Gabriele S. Kaminski, additional
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- 2020
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25. The role of water in amyloid aggregation kinetics
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Stephens, Amberley D, primary and Kaminski Schierle, Gabriele S, additional
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- 2019
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26. Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity
- Author
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Stephens, Amberley D., primary, Zacharopoulou, Maria, additional, Moons, Rani, additional, Fusco, Giuliana, additional, Seetaloo, Neeleema, additional, Chiki, Anass, additional, Hooper, Philippa J., additional, Mela, Ioanna, additional, Lashuel, Hilal A., additional, Phillips, Jonathan J, additional, Simone, Alfonso De, additional, Sobott, Frank, additional, and Schierle, Gabriele S. Kaminski, additional
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- 2019
- Full Text
- View/download PDF
27. Different structural conformers of monomeric alpha-synuclein identified after lyophilising and freezing
- Author
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Stephens, Amberley D, Nespovitaya, Nadezhda, Zacharopoulou, Maria, Kaminski, Clemens, Phillips, Jonathan, Kaminski, GS, Zacharopoulou, Maria [0000-0002-3660-2797], Kaminski, Clemens [0000-0002-5194-0962], Kaminski Schierle, Gabriele [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Protein Conformation ,Freezing ,alpha-Synuclein ,Humans ,Parkinson Disease - Abstract
Understanding the mechanisms behind amyloid protein aggregation in diseases such as Parkinson’s and Alzheimer’s disease is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein alpha-synuclein (aSyn), a protein involved in Parkinson’s disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods including, size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterise the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterise the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilising the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD., G.S.K.S. and C.F.K. acknowledge funding from the Wellcome Trust, the UK Medical Research Council (MRC), Alzheimer Research UK (ARUK), and Infinitus China Ltd. C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (EPSRC). M.Z. acknowledges funding from the Eugenides Foundation, Newnham College (Cambridge), and George and Marie Vergottis Foundation (Cambridge Trust). A.D.S. and M.Z. acknowledge the European Biophysical Societies’ Association (EBSA), Alzheimer Research UK (ARUK), Newnham College (Cambridge) and British Mass Spectrometry Society (BMSS) for travel grants.
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- 2018
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28. Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans
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Huang, Chaolie, primary, Wagner-Valladolid, Sara, additional, Stephens, Amberley D, additional, Jung, Raimund, additional, Poudel, Chetan, additional, Sinnige, Tessa, additional, Lechler, Marie C, additional, Schlörit, Nicole, additional, Lu, Meng, additional, Laine, Romain F, additional, Michel, Claire H, additional, Vendruscolo, Michele, additional, Kaminski, Clemens F, additional, Kaminski Schierle, Gabriele S, additional, and David, Della C, additional
- Published
- 2019
- Full Text
- View/download PDF
29. The Cellular Environment Affects Monomeric α-Synuclein Structure
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Stephens, Amberley D., primary, Zacharopoulou, Maria, additional, and Kaminski Schierle, Gabriele S., additional
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- 2019
- Full Text
- View/download PDF
30. Author response: Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans
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Huang, Chaolie, primary, Wagner-Valladolid, Sara, additional, Stephens, Amberley D, additional, Jung, Raimund, additional, Poudel, Chetan, additional, Sinnige, Tessa, additional, Lechler, Marie C, additional, Schlörit, Nicole, additional, Lu, Meng, additional, Laine, Romain F, additional, Michel, Claire H, additional, Vendruscolo, Michele, additional, Kaminski, Clemens F, additional, Kaminski Schierle, Gabriele S, additional, and David, Della C, additional
- Published
- 2019
- Full Text
- View/download PDF
31. Intra-mitochondrial proteostasis is directly coupled to alpha-synuclein and Amyloid β 1-42 pathology
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Lautenschläger, Janin, primary, Wagner-Valladolid, Sara, additional, Stephens, Amberley D., additional, Fernández-Villegas, Ana, additional, Hockings, Colin, additional, Mishra, Ajay, additional, Manton, James D., additional, Fantham, Marcus J., additional, Lu, Meng, additional, Rees, Eric J., additional, Kaminski, Clemens F., additional, and Schierle, Gabriele S. Kaminski, additional
- Published
- 2019
- Full Text
- View/download PDF
32. Low energy optical excitations as an indicator of structural changes initiated at the termini of amyloid proteins
- Author
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Jong, Kwang Hyok, primary, Azar, Yavar T., additional, Grisanti, Luca, additional, Stephens, Amberley D., additional, Jones, Saul T. E., additional, Credgington, Dan, additional, Kaminski Schierle, Gabriele S., additional, and Hassanali, Ali, additional
- Published
- 2019
- Full Text
- View/download PDF
33. Structural basis of synaptic vesicle assembly promoted by α-synuclein
- Author
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Fusco, Giuliana, Pape, Tillmann, Stephens, Amberley D, Mahou, Pierre, Costa, Ana Rita, Kaminski, Clemens F, Kaminski Schierle, Gabriele S, Vendruscolo, Michele, Veglia, Gianluigi, Dobson, Christopher M, De Simone, Alfonso, Fusco, G., Pape, T., Stephens, A. D., Mahou, P., Costa, A. R., Kaminski, C. F., Kaminski Schierle, G. S., Vendruscolo, M., Veglia, G., Dobson, C. M., De Simone, A., Kaminski, Clemens [0000-0002-5194-0962], Kaminski Schierle, Gabriele [0000-0002-1843-2202], Vendruscolo, Michele [0000-0002-3616-1610], Apollo - University of Cambridge Repository, and Medical Research Council (MRC)
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SOLUTION NMR-SPECTROSCOPY ,MEMBRANE INTERACTIONS ,animal diseases ,Science ,PROTEIN ,Molecular Dynamics Simulation ,PARKINSONS-DISEASE ,PHOSPHOLIPID-BINDING ,Escherichia coli ,Animals ,SATURATION-TRANSFER DIFFERENCE ,Science & Technology ,Cell Membrane ,FLUORESCENCE MICROSCOPY ,IN-VITRO ,Corrigenda ,SOLID-STATE NMR ,Rats ,nervous system diseases ,Multidisciplinary Sciences ,nervous system ,N-TERMINAL ACETYLATION ,alpha-Synuclein ,Science & Technology - Other Topics ,Synaptic Vesicles - Abstract
α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson’s disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson’s disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this ‘double-anchor’ mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts.
- Published
- 2016
34. Structural basis of synaptic vesicle assembly promoted by α-synuclein
- Author
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Fusco, Giuliana, primary, Pape, Tillmann, additional, Stephens, Amberley D., additional, Mahou, Pierre, additional, Costa, Ana Rita, additional, Kaminski, Clemens F., additional, Kaminski Schierle, Gabriele S., additional, Vendruscolo, Michele, additional, Veglia, Gianluigi, additional, Dobson, Christopher M., additional, and De Simone, Alfonso, additional
- Published
- 2016
- Full Text
- View/download PDF
35. C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction
- Author
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Lautenschläger, Janin, Stephens, Amberley D, Fusco, Giuliana, Ströhl, Florian, Curry, Nathan, Zacharopoulou, Maria, Michel, Claire H, Laine, Romain, Nespovitaya, Nadezhda, Fantham, Marcus, Pinotsi, Dorothea, Zago, Wagner, Fraser, Paul, Tandon, Anurag, St George-Hyslop, Peter, Rees, Eric, Phillips, Jonathan J, De Simone, Alfonso, Kaminski, Clemens F, and Schierle, Gabriele S Kaminski
- Subjects
Binding Sites ,animal diseases ,Presynaptic Terminals ,In Vitro Techniques ,Lipid Metabolism ,nervous system diseases ,3. Good health ,Cell Line ,Rats ,Rats, Sprague-Dawley ,Protein Aggregates ,nervous system ,Microscopy, Electron, Transmission ,alpha-Synuclein ,Animals ,Humans ,Calcium ,Synaptic Vesicles ,Nuclear Magnetic Resonance, Biomolecular ,Protein Binding ,Synaptosomes - Abstract
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
36. C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction
- Author
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Lautenschläger, Janin, Stephens, Amberley D., Fusco, Giuliana, Ströhl, Florian, Curry, Nathan, Zacharopoulou, Maria, Michel, Claire H., Laine, Romain, Nespovitaya, Nadezhda, Fantham, Marcus, Pinotsi, Dorothea, Zago, Wagner, Fraser, Paul, Tandon, Anurag, St George-Hyslop, Peter, Rees, Eric, Phillips, Jonathan J., De Simone, Alfonso, Kaminski, Clemens F., and Kaminski Schierle, Gabriele S.
- Subjects
nervous system ,animal diseases ,nervous system diseases ,3. Good health - Abstract
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies., Nature Communications, 9, ISSN:2041-1723
37. Fast Purification of Recombinant Monomeric Amyloid-β from E. coli and Amyloid-β-mCherry Aggregates from Mammalian Cells
- Author
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Stephens, Amberley D, Lu, Meng, Fernandez-Villegas, Ana, and Kaminski Schierle, Gabriele S
- Subjects
Amyloid beta-Peptides ,Aβ42 ,amyloid ,dye labeling ,inclusion bodies ,ion exchange chromatography ,mCherry ,Aβ40 ,maleimide ,Peptide Fragments ,Recombinant Proteins ,3. Good health ,Alzheimer Disease ,Escherichia coli ,Animals ,E22G ,arctic mutant ,fluorescence - Abstract
The Alzheimer's disease related peptide, Amyloid-beta (Aβ)1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction, or the need to reconstitute Aβ from a freeze-dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The washing, solubilization, and purification steps take only 3 h. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.
38. Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies
- Author
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Lautenschläger, Janin, Wagner-Valladolid, Sara, Stephens, Amberley D, Fernández-Villegas, Ana, Hockings, Colin, Mishra, Ajay, Manton, James D, Fantham, Marcus J, Lu, Meng, Rees, Eric J, Kaminski, Clemens F, and Kaminski Schierle, Gabriele S
- Subjects
amyloid-β (Aβ,) ,HtrA2/Omi ,Nerve Tissue Proteins ,protein aggregation ,Rats, Sprague-Dawley ,neurodegenerative disease ,α-synuclein ,amyloid-β (AB) ,Cell Line, Tumor ,Animals ,Humans ,Lon peptidase 1 mitochondrial ,protein homeostasis ,Amyloid beta-Peptides ,Serine-Arginine Splicing Factors ,neurodegeneration ,HtrA serine peptidase 2 ,Lon protease ,Parkinson Disease ,High-Temperature Requirement A Serine Peptidase 2 ,Peptide Fragments ,3. Good health ,Mitochondria ,Rats ,α-synuclein (a-synuclein) ,Proteostasis ,alpha-Synuclein ,Female - Abstract
Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.
39. Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
- Author
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Stephens, Amberley D., Zacharopoulou, Maria, Moons, Rani, Fusco, Giuliana, Seetaloo, Neeleema, Chiki, Anass, Woodhams, Philippa J., Mela, Ioanna, Lashuel, Hilal A., Phillips, Jonathan J., De Simone, Alfonso, Sobott, Frank, and Schierle, Gabriele S. Kaminski
- Subjects
82/80 ,82/58 ,692/699/375/365/1718 ,140/131 ,article ,631/45/535/878 ,631/57/2269 ,82/83 ,631/1647/296 ,3. Good health - Abstract
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn’s aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
40. Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
- Author
-
Stephens, Amberley D, Zacharopoulou, Maria, Moons, Rani, Fusco, Giuliana, Seetaloo, Neeleema, Chiki, Anass, Woodhams, Philippa J, Mela, Ioanna, Lashuel, Hilal A, Phillips, Jonathan J, De Simone, Alfonso, Sobott, Frank, and Schierle, Gabriele S Kaminski
- Subjects
Kinetics ,Protein Aggregates ,Structure-Activity Relationship ,Protein Conformation ,Proton Magnetic Resonance Spectroscopy ,Mutation ,alpha-Synuclein ,Humans ,Calcium ,Mutant Proteins ,Benzothiazoles ,Phosphorylation ,3. Good health - Abstract
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.
41. Corrigendum: Structural basis of synaptic vesicle assembly promoted by α-synuclein.
- Author
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Fusco, Giuliana, Pape, Tillmann, Stephens, Amberley D., Mahou, Pierre, Costa, Ana Rita, Kaminski, Clemens F., Schierle, Gabriele S. Kaminski, Vendruscolo, Michele, Veglia, Gianluigi, Dobson, Christopher M., and De Simone, Alfonso
- Published
- 2017
- Full Text
- View/download PDF
42. Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity
- Author
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Alfonso De Simone, Ioanna Mela, Giuliana Fusco, Frank Sobott, Rani Moons, Jonathan J. Phillips, Anass Chiki, Philippa J. Woodhams, Maria Zacharopoulou, Neeleema Seetaloo, Gabriele S. Kaminski Schierle, Hilal A. Lashuel, Amberley D. Stephens, Stephens, Amberley D [0000-0002-7303-6392], Schierle, Gabriele S Kaminski [0000-0002-1843-2202], Apollo - University of Cambridge Repository, Stephens, A. D., Zacharopoulou, M., Moons, R., Fusco, G., Seetaloo, N., Chiki, A., Woodhams, P. J., Mela, I., Lashuel, H. A., Phillips, J. J., De Simone, A., Sobott, F., Schierle, G. S. K., Stephens, Amberley D. [0000-0002-7303-6392], and Schierle, Gabriele S. Kaminski [0000-0002-1843-2202]
- Subjects
0301 basic medicine ,binding ,Protein Conformation ,Parkinson's disease ,Proton Magnetic Resonance Spectroscopy ,692/699/375/365/1718 ,General Physics and Astronomy ,Sequence (biology) ,Protein aggregation ,631/45/535/878 ,chemistry.chemical_compound ,0302 clinical medicine ,Protein structure ,Mutant Protein ,Phosphorylation ,lcsh:Science ,health care economics and organizations ,Multidisciplinary ,dynamics ,3. Good health ,Monomer ,140/131 ,alpha-Synuclein ,631/57/2269 ,Engineering sciences. Technology ,631/1647/296 ,Human ,Science ,General Biochemistry, Genetics and Molecular Biology ,Article ,82/80 ,03 medical and health sciences ,Protein Aggregates ,Structure-Activity Relationship ,NMR spectroscopy ,Structure–activity relationship ,Humans ,Benzothiazoles ,fibrillation ,82/83 ,Alpha-synuclein ,wild-type ,Kinetic ,Intrinsically disordered proteins ,Mass spectrometry ,82/58 ,Wild type ,General Chemistry ,Benzothiazole ,proteins ,Kinetics ,030104 developmental biology ,chemistry ,parkinson ,Mutation ,Biophysics ,residual structure ,Calcium ,Mutant Proteins ,lcsh:Q ,heterogeneity ,030217 neurology & neurosurgery - Abstract
As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn’s aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein., In Parkinson’s disease (PD) the monomeric protein alpha-synuclein (aSyn) misfolds and aggregates into insoluble fibrils. Here the authors use NMR measurements and hydrogen–deuterium exchange mass spectrometry and find that the more solvent exposed the N-terminus of aSyn is, the more aggregation prone its conformation becomes, and further show how PD mutations and post translational modifications influence the extent of the N-terminus solvent exposure.
- Published
- 2020
- Full Text
- View/download PDF
43. Decreased Water Mobility Contributes To Increased α-Synuclein Aggregation
- Author
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Amberley D. Stephens, Johanna Kölbel, Rani Moons, Chyi Wei Chung, Michael T. Ruggiero, Najet Mahmoudi, Talia A. Shmool, Thomas M. McCoy, Daniel Nietlispach, Alexander F. Routh, Frank Sobott, J. Axel Zeitler, Gabriele S. Kaminski Schierle, Stephens, Amberley D [0000-0002-7303-6392], Kölbel, Johanna [0000-0002-9820-1892], Moons, Rani [0000-0003-2961-9950], Chung, Chyi Wei [0000-0003-1780-3486], Ruggiero, Michael T [0000-0003-1848-2565], Mahmoudi, Najet [0000-0003-4936-6911], Shmool, Talia A [0000-0002-0415-3050], McCoy, Thomas M [0000-0002-4897-7924], Nietlispach, Daniel [0000-0003-4364-9291], Routh, Alexander F [0000-0002-3443-3053], Sobott, Frank [0000-0001-9029-1865], Zeitler, J Axel [0000-0002-4958-0582], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], Apollo - University of Cambridge Repository, and Routh, Alex [0000-0002-3443-3053]
- Subjects
Amyloid ,Solvation Shell ,34 Chemical Sciences ,Forschungsartikel ,Water ,Parkinson Disease ,General Medicine ,General Chemistry ,Catalysis ,Hydration Shell ,Chemistry ,Hydrogen Bond ,Solvent ,3406 Physical Chemistry ,alpha-Synuclein ,Solvents ,Humans - Abstract
The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H2O molecules influences the aggregation rate of the amyloid protein alpha-synuclein (alpha Syn), a protein associated with Parkinson's disease. When the mobility of H2O within the solvation shell is reduced by the presence of NaCl, alpha Syn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and alpha Syn aggregation is reduced. Changing the solvent from H2O to D2O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of alpha Syn, it is also influenced by a reduction in both the H2O mobility and alpha Syn mobility. We propose that reduced mobility of alpha Syn contributes to increased aggregation by promoting intermolecular interactions.
- Published
- 2023
- Full Text
- View/download PDF
44. Intracellular Aβ42 Aggregation Leads to Cellular Thermogenesis
- Author
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Chyi Wei Chung, Amberley D. Stephens, Tasuku Konno, Edward Ward, Edward Avezov, Clemens F. Kaminski, Ali A. Hassanali, Gabriele S. Kaminski Schierle, Chung, Chyi Wei [0000-0003-1780-3486], Stephens, Amberley D [0000-0002-7303-6392], Kaminski, Clemens F [0000-0002-5194-0962], Hassanali, Ali A [0000-0002-3208-1488], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Colloid and Surface Chemistry ,Amyloid beta-Peptides ,Alzheimer Disease ,Data_MISCELLANEOUS ,Humans ,Thermogenesis ,General Chemistry ,Biochemistry ,ComputingMilieux_MISCELLANEOUS ,Catalysis ,Peptide Fragments - Abstract
Funder: Cambridge Commonwealth, European and International Trust, Funder: Infinitus China Ltd., The aggregation of Aβ42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aβ42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aβ42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aβ42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aβ peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aβ peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.
- Published
- 2022
45. Fast Purification of Recombinant Monomeric Amyloid-β from E. coli and Amyloid-β-mCherry Aggregates from Mammalian Cells
- Author
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Ana Fernández-Villegas, Meng Lu, Amberley D. Stephens, Gabriele S. Kaminski Schierle, Stephens, Amberley D [0000-0002-7303-6392], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Amyloid ,Physiology ,Cognitive Neuroscience ,Ion chromatography ,inclusion bodies ,Peptide ,Biochemistry ,Inclusion bodies ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Alzheimer Disease ,law ,Escherichia coli ,Animals ,E22G ,MTT assay ,arctic mutant ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Amyloid beta-Peptides ,Chemistry ,Aβ42 ,amyloid ,dye labeling ,ion exchange chromatography ,mCherry ,Cell Biology ,General Medicine ,Aβ40 ,maleimide ,Peptide Fragments ,Recombinant Proteins ,3. Good health ,Monomer ,Recombinant DNA ,fluorescence ,030217 neurology & neurosurgery - Abstract
The Alzheimer's disease related peptide, Amyloid-beta (Aβ)1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of Aβ isolation, the formation of Aβ multimers within the time frame of extraction, or the need to reconstitute Aβ from a freeze-dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant Aβ using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The washing, solubilization, and purification steps take only 3 h. We also present a protocol for the isolation of Aβ-mCherry from mammalian cells.
- Published
- 2020
46. Label-free characterisation of amyloids and alpha-Synuclein polymorphs by exploiting their intrinsic fluorescence property
- Author
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Gabriele S. Kaminski Schierle, Edward Ward, Clemens F. Kaminski, Yuqing Feng, Molly Jo Davis, Chyi Wei Chung, Amberley D. Stephens, Chung, Chyi Wei [0000-0003-1780-3486], Stephens, Amberley D [0000-0002-7303-6392], Kaminski, Clemens F [0000-0002-5194-0962], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Alpha-synuclein ,Amyloid ,Chemistry ,Amyloidogenic Proteins ,Intrinsic fluorescence ,Fibril ,Fluorescence ,Small molecule ,In vitro ,Analytical Chemistry ,chemistry.chemical_compound ,alpha-Synuclein ,Biophysics ,Protein Conformation, beta-Strand ,Label free - Abstract
Funder: Infinitus China Ltd., Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.
- Published
- 2021
47. Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies
- Author
-
Ana Fernández-Villegas, Gabriele S. Kaminski Schierle, Marcus Fantham, Janin Lautenschläger, Amberley D. Stephens, Sara Wagner-Valladolid, James D. Manton, Eric J. Rees, Colin Hockings, Ajay Kumar Mishra, Clemens F. Kaminski, Meng Lu, Lautenschläger, Janin [0000-0001-7788-7074], Stephens, Amberley D [0000-0002-7303-6392], Manton, James D [0000-0001-9260-3156], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Proteases ,amyloid-β (Aβ,) ,Amyloid ,HtrA2/Omi ,Nerve Tissue Proteins ,Mitochondrion ,Protein aggregation ,Biochemistry ,protein aggregation ,Rats, Sprague-Dawley ,03 medical and health sciences ,α-synuclein ,neurodegenerative disease ,amyloid-β (AB) ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Molecular Biology ,protein homeostasis ,Lon peptidase 1 mitochondrial ,Amyloid beta-Peptides ,030102 biochemistry & molecular biology ,Serine-Arginine Splicing Factors ,Chemistry ,Neurodegeneration ,neurodegeneration ,HtrA serine peptidase 2 ,Lon protease ,Molecular Bases of Disease ,Parkinson Disease ,Cell Biology ,High-Temperature Requirement A Serine Peptidase 2 ,medicine.disease ,Peptide Fragments ,3. Good health ,Cell biology ,Mitochondria ,Rats ,α-synuclein (a-synuclein) ,Cytosol ,030104 developmental biology ,Proteostasis ,alpha-Synuclein ,Female - Abstract
Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.
- Published
- 2020
48. Short hydrogen bonds enhance nonaromatic protein-related fluorescence
- Author
-
J. Axel Zeitler, Philippa J. Woodhams, Gabriele S. Kaminski Schierle, Gonzalo Diaz Miron, Amberley D. Stephens, Michael T. Ruggiero, Elyse M. Kleist, Uriel N. Morzan, Ali Hassanali, Luca Grisanti, Dan Credgington, Andrew D. Bond, Saul T. E. Jones, Muhammad Nawaz Qaisrani, Ralph Gebauer, Mariano C. González Lebrero, Emiliano Poli, Stephens, Amberley D [0000-0002-7303-6392], Qaisrani, Muhammad Nawaz [0000-0003-4167-3116], Ruggiero, Michael T [0000-0003-1848-2565], Woodhams, Philippa J [0000-0003-4537-5976], Kleist, Elyse M [0000-0001-8596-659X], Zeitler, J Axel [0000-0002-4958-0582], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Chemical transformation ,Optics and Photonics ,Glutamine ,Intrinsic fluorescence ,Molecular Dynamics Simulation ,Photochemistry ,Fluorescence ,Ab initio molecular dynamics ,Ammonia ,Humans ,Single amino acid ,short hydrogen bond ,Density Functional Theory ,Multidisciplinary ,Hydrogen bond ,Chemistry ,intrinsic fluorescence ,ultraviolet fluorescence ,Hydrogen Bonding ,Conical intersection ,Biophysics and Computational Biology ,Excited state ,Physical Sciences ,Peptides - Abstract
Significance Intrinsic fluorescence of nonaromatic amino acids is a puzzling phenomenon with an enormous potential in biophotonic applications. The physical origins of this effect, however, remain elusive. Herein, we demonstrate how specific hydrogen bond networks can modulate fluorescence. We highlight the key role played by short hydrogen bonds, present in the protein structure, on the ensuing fluorescence. We provide detailed experimental and molecular evidence to explain these unusual nonaromatic optical properties. Our findings should benefit the design of novel optically active biomaterials for applications in biosensing and imaging., Fluorescence in biological systems is usually associated with the presence of aromatic groups. Here, by employing a combined experimental and computational approach, we show that specific hydrogen bond networks can significantly affect fluorescence. In particular, we reveal that the single amino acid L-glutamine, by undergoing a chemical transformation leading to the formation of a short hydrogen bond, displays optical properties that are significantly enhanced compared with L-glutamine itself. Ab initio molecular dynamics simulations highlight that these short hydrogen bonds prevent the appearance of a conical intersection between the excited and the ground states and thereby significantly decrease nonradiative transition probabilities. Our findings open the door to the design of new photoactive materials with biophotonic applications.
- Published
- 2021
49. Purification of Recombinant α-synuclein: A Comparison of Commonly Used Protocols
- Author
-
Gabriele S. Kaminski Schierle, Dijana Matak-Vinkovic, Ana Fernández-Villegas, Amberley D. Stephens, Stephens, Amberley D [0000-0002-7303-6392], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], and Apollo - University of Cambridge Repository
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Lysis ,Cell Survival ,Protein Conformation ,Electrospray ionization ,Ion chromatography ,Size-exclusion chromatography ,Protein aggregation ,Biochemistry ,Article ,Cell Line ,03 medical and health sciences ,Protein Aggregates ,Protein structure ,Microscopy, Electron, Transmission ,Escherichia coli ,Chemical Precipitation ,Humans ,Ammonium sulfate precipitation ,0303 health sciences ,Chemistry ,030302 biochemistry & molecular biology ,Periplasmic space ,Chromatography, Ion Exchange ,Recombinant Proteins ,Intrinsically Disordered Proteins ,Chromatography, Gel ,alpha-Synuclein ,Protein Conformation, beta-Strand ,Chromatography, Liquid - Abstract
The initial state of the intrinsically disordered protein α-synuclein (aSyn), e.g., the presence of oligomers and degradation products, or the presence of contaminants and adducts can greatly influence the aggregation kinetics and toxicity of the protein. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from Escherichia coli: boiling, acid precipitation, ammonium sulfate precipitation, and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using nondenaturing electrospray ionization mass spectrometry, that aSyn isolated by acid precipitation and periplasmic lysis was the purest and yielded the highest percentage of monomeric protein, 100% and 96.5%, respectively. We then show that aSyn purified by the different protocols exerts different metabolic stresses in cells, with the more multimeric/degraded and least pure samples leading to a larger increase in cell vitality. However, the percentage of monomeric protein and the purity of the samples did not correlate with aSyn aggregation propensity. This study highlights the importance of characterizing monomeric aSyn after purification, as the choice of purification method can significantly influence the outcome of a subsequent study.
- Published
- 2020
50. Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans
- Author
-
Chaolie Huang, Michele Vendruscolo, Amberley D. Stephens, Marie C. Lechler, Nicole Schlörit, Clemens F. Kaminski, Tessa Sinnige, Romain F. Laine, Claire H. Michel, Chetan Poudel, Sara Wagner-Valladolid, Della C. David, Meng Lu, Raimund Jung, Gabriele S. Kaminski Schierle, Stephens, Amberley D [0000-0002-7303-6392], Sinnige, Tessa [0000-0002-9353-126X], Lu, Meng [0000-0001-9311-2666], Laine, Romain F [0000-0002-2151-4487], Vendruscolo, Michele [0000-0002-3616-1610], Kaminski, Clemens F [0000-0002-5194-0962], Kaminski Schierle, Gabriele S [0000-0002-1843-2202], David, Della C [0000-0001-8597-9470], and Apollo - University of Cambridge Repository
- Subjects
metabolism [Caenorhabditis elegans Proteins] ,0301 basic medicine ,Aging ,Amyloid ,QH301-705.5 ,Science ,Structural Biology and Molecular Biophysics ,Normal aging ,physiology [Caenorhabditis elegans] ,Protein aggregation ,General Biochemistry, Genetics and Molecular Biology ,protein aggregation ,03 medical and health sciences ,Protein Aggregates ,0302 clinical medicine ,cell biology ,molecular biophysics ,structural biology ,Animals ,Biology (General) ,Functional decline ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Amyloid like ,metabolism [Amyloid] ,General Immunology and Microbiology ,biology ,Chemistry ,General Neuroscience ,General Medicine ,Amyloid fibril ,biology.organism_classification ,Cell biology ,030104 developmental biology ,Structural biology ,C. elegans ,Medicine ,ddc:600 ,030217 neurology & neurosurgery ,Research Article - Abstract
Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life.
- Published
- 2019
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