Synuclein plasticity: the Achilles’ heel of nerve function linked to the onset of Parkinson’s disease

Lewy bodies – the hallmarks of Parkinson’s disease – are majorly constituted of aggregates of the presynaptic protein alpha-synuclein. The molecular mechanism of alpha-synuclein aggregation through which it changes dramatically from a soluble disordered monomer to insoluble structured fibrils remains unknown. As an intrinsically disordered protein, alpha-synuclein does not have a specific three-dimensional structure, but rather behaves mostly as a meta-stable ensemble of highly dynamic conformers, and as such undergoes rapid kinetics, making it almost impossible to measure its conformational changes with most techniques. Millisecond amide hydrogen exchange can provide valuable insights on the dynamic behaviour of proteins, especially at flexible regions. Thus, the work in this thesis reports on the development of methods and tools for hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and the application of these for the study of aSyn under physiological conditions. In the first part of this thesis, high resolution on the alpha-synuclein monomer was achieved over two dimensions: time and space. Using a novel in-house rapid- mixing quench-flow instrument, hydrogen/deuterium-exchange mass spectrometry data on alpha-synuclein on the millisecond timescale was attained. Furthermore, using a ‘soft’ gas-phase mass spectrometry fragmentation technique called Electron Transfer Dissociation, structural resolution in the protein increased. The second part of this work focuses on the development of a software, HDfleX, in an effort to primarily automate the HDX-MS workflow and allow the merging of HDX-MS data at different levels: bottom-up, middle-down and top-down. The rest of the thesis uses the tools and methods developed earlier on to explore the effects of different solution conditions (cellular compartments and salt cations) on the monomeric conformations of aSyn, and how these correlate to the different stages of aggregation and the ensuing fibril polymorphs. Altogether, the achievements in this work will allow us to better understand the plasticity of the alpha-synuclein monomer as it cycles through different local environments

Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity

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

Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity

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

Back to the Roots : Revisiting the Use of the Fiber-Rich Cichorium intybus L. Taproots

Fibers are increasingly recognized as an indispensable part of our diet and vital for maintaining health. Notably, complex mixtures of fibers have been found to improve metabolic health. Following an analysis of the fiber content of plant-based products, we found the taproot of the chicory plant (Cichorium intybus L) to be 1 of the vegetables with the highest fiber content, comprising nearly 90% of its dry weight. Chicory roots consist of a mixture of inulin, pectin, and (hemi-)cellulose and also contain complex phytochemicals, such as sesquiterpene lactones that have been characterized in detail. Nowaday, chicory roots are mainly applied as a source for the extraction of inulin, which is used as prebiotic fiber and food ingredient. Chicory roots, however, have long been consumed as a vegetable by humans. The whole root has been used for thousands of years for nutritional, medicinal, and other purposes, and it is still used in traditional dishes in various parts of the world. Here, we summarize the composition of chicory roots to explain their historic success in the human diet. We revisit the intake of chicory roots by humans and describe the different types of use along with their various methods of preparation. Hereby, we focus on the whole root in its complex, natural form, as well as in relation to its constituents, and discuss aspects regarding legal regulation and the safety of chicory root extracts for human consumption. Finally, we provide an overview of the current and future applications of chicory roots and their contribution to a fiber-rich diet.Peer reviewe

Millisecond hydrogen/deuterium-exchange mass spectrometry for the study of alpha-synuclein structural dynamics under physiological conditions

This is the author accepted manuscript. The final version is available from MyJove Corporation via the DOI in this recordAlpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and thestructural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.UK Research and InnovationInnovate U

HDfleX: Software for the flexible structural resolution of hydrogen-deuterium exchange mass spectrometry data

All rights reserved. For academic and research purposes only. If interested in a commercial license, please contact [email protected]. Tutorial videos can be found at: https://www.youtube.com/channel/UCroMi29q1tTZfNJ-qM8ADNAThe article associated with this software is available in ORE at: http://hdl.handle.net/10871/129204A standalone application for the post-processing of hydrogen-deuterium exchange mass spectrometry data, including curve fitting, merging of bottom-up and middle-down data and robust statistical significance analyses, amongst others. The scientific basis and application of HDfleX has been described thoroughly in the following publication: Neeleema Seetaloo, Monika Kish, and Jonathan J. Phillips. Analytical Chemistry Article ASAP. DOI: 10.1021/acs.analchem.1c053391.

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein.

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

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α‑Synuclein

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

Research data supporting 'Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity'

Raw data files supporting the manuscript 'Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity'. Including raw HDX-MS data for up to 6 replicates for two labelling conditions (with and without Ca2+) and MS/MS mapping files. Raw nano-ESI-EM and nano-ESI-IM-MS data of three replicates with and without Ca2+. Raw NMR data files for three aSyn varients with and without Ca2+. Raw ThT-based aggregation kinetic data. Additional AFM files. Primer sequences