32 research outputs found

    Phylogeographic diversity and mosaicism of the Helicobacter pylori tfs integrative and conjugative elements.

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    Background: The genome of the gastric pathogen Helicobacter pylori is characterised by considerable variation of both gene sequence and content, much of which is contained within three large genomic islands comprising the cag pathogenicity island (cagPAI) and two mobile integrative and conjugative elements (ICEs) termed tfs3 and tfs4. All three islands are implicated as virulence factors, although whereas the cagPAI is well characterised, understanding of how the tfs elements influence H. pylori interactions with different human hosts is significantly confounded by limited definition of their distribution, diversity and structural representation in the global H. pylori population. Results: To gain a global perspective of tfs ICE population dynamics we established a bioinformatics workflow to extract and precisely define the full tfs pan-gene content contained within a global collection of 221 draft and complete H. pylori genome sequences. Complete (ca. 35-55kbp) and remnant tfs ICE clusters were reconstructed from a dataset comprising >12,000 genes, from which orthologous gene complements and distinct alleles descriptive of different tfs ICE types were defined and classified in comparative analyses. The genetic variation within defined ICE modular segments was subsequently used to provide a complete description of tfs ICE diversity and a comprehensive assessment of their phylogeographic context. Our further examination of the apparent ICE modular types identified an ancient and complex history of ICE residence, mobility and interaction within particular H. pylori phylogeographic lineages and further, provided evidence of both contemporary inter-lineage and inter-species ICE transfer and displacement. Conclusions: Our collective results establish a clear view of tfs ICE diversity and phylogeographic representation in the global H. pylori population, and provide a robust contextual framework for elucidating the functional role of the tfs ICEs particularly as it relates to the risk of gastric disease associated with different tfs ICE genotypes

    Structural basis of synaptic vesicle assembly promoted by α-synuclein.

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    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

    α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons

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    Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson’s disease patient’s brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons

    Short hydrogen bonds enhance nonaromatic protein-related fluorescence.

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    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

    C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.

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    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

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

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    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
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