Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.
Summary Filamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. Interestingly, although degradation of monomeric tau is not inhibited by adenosine 5’-(3-thiotriphosphate) (ATPγS), fibril fragmentation is predominantly dependent on the ATPase activity of the proteasome. The proteasome holoenzyme also targets fibrils assembled from α-synuclein, suggesting that its fibril-fragmenting function may be a general mechanism. The fragmented species produced by the proteasome shows significant toxicity to human cell lines compared with intact fibrils. Together, our results indicate that the proteasome holoenzyme possesses a fragmentation function that disassembles large fibrils into smaller and more cytotoxic species.
Filamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Although there have been numerous studies of the aggregation process in vitro, far fewer studies of aggregate disassembly and degradation are available. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. TEM further revealed that these small aggregate species had no distinct structure. The intact proteasome holoenzyme is required to effectively target fibrils. Interestingly, while degradation of monomeric tau was not inhibited by ATPγS, fibril fragmentation was predominantly dependent on the ATPase activity of the proteasome. The proteasome holoenzyme was also found to target fibrils assembled from α -synuclein (αS), suggesting that its fibril fragmenting function may be a general mechanism. The fragmented species produced by the proteasome showed significant toxicity to human cell lines compared to intact fibrils. Together, our results indicate that the proteasome holoenzyme possesses a novel fragmentation function that disassembles large fibrils into smaller and more cytotoxic species. (196 words)
oligomers of pneumolysin form transmembrane channels in cholesterol-containing lipid bilayers. the mechanism of pore formation involves a multistage process in which the protein, at first, assembles into a ring-shaped complex on the outer-bilayer leaflet. In a subsequent step, the complex inserts into the membrane. contrary to most investigations of pore formation that have focussed on protein changes, we have deduced how the lipid-packing order is altered in different stages of the poreforming mechanism. An optical tweezing apparatus was used, in combination with microfluidics, to isolate large-unilamellar vesicles and control exposure of the bilayer to pneumolysin. By monitoring Raman-scattered light from a single-trapped liposome, the effect of the protein on short-range order and rotational diffusion of lipids could be inferred from changes in the envelope of the C-H stretch. A significant change in the lipid-packing order takes place during assembly of pre-pore oligomers. We were not able to detect a change in the lipid-packing order during the initial stage of protein binding, or any further change during the insertion of oligomers. pre-pore complexes induce a transformation in which a bilayer, resembling a liquid-ordered phase is changed into a bilayer resembling a fluid-liquiddisordered phase surrounding ordered microdomains enriched in cholesterol and protein complexes. Cholesterol-dependent cytolysins (CDCs) are a family of more than 20 cytolytic proteins that are highly conserved. A representative member of this family is pneumolysin (PLY), a major virulence factor of the bacterium Streptococcus pneumoniae 1 , which is responsible for a wide range of infectious diseases, including pneumonia, bronchitis, meningitis, sepsis and otitis media 2. These cytolysins, along with the membrane attack complex of complement and perforin 3 , are believed to share a common mechanism for pore formation in lipid bilayers. Structural data have been obtained by cryo-electron microscopy (cryo-EM) showing that oligomeric complexes of PLY, with up to 44 subunits 4 , form transmembrane channels with a diameter of 26 nm 5. This prior work has also confirmed the multistage nature of the interaction between CDC subunits, in which the self-assembly of a pre-pore complex on the outer-bilayer leaflet takes place before a concerted change in the conformational structure of monomer subunits that leads to insertion into membranes. In more recent work, both cryo-EM and real-time atomic-force microscopy have been used to visualise individual steps in the assembly pathway of a related CDC, suilysin 6. PLY, and other CDCs, have four domains, a membrane attachment domain 4 (D4) and three noncontiguous domains, D1-D3. The soluble form of PLY commonly exists as monomers 7,8. Large circular structures form at higher concentrations 8,9 and X-ray crystallography has shown that PLY forms linear oligomers in the solid state 9. Domain 4 is the membrane binding domain and the interaction enables a tryptophan loop at the base of the
17Neurodegenerative diseases such as Alzheimer′s and Parkinson′s are associated with protein 18 misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous 19 oligomeric species, formed early on in the aggregation process, may be a source of 20 cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study 21 of amyloid proteins and aggregation processes. However, the poor photophysical and binding 22properties of ThT impairs the study of oligomers. To overcome this challenge, we have 23 designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior 24 properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to 25 amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural 26 amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used 27 as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, we 28 demonstrate that ThX can be used to detect a distinct oligomeric species, not observed via 29 traditional ThT imaging.30 31 157 as cerebrospinal fluid and blood plasma. 158 159 Contributions 160 synthesized the molecules. J.W. and L.M.N. performed bulk fluorescence characterisation 162 measurements and analyses. J.W. prepared the αSyn aggregates, performed bulk protein 163 binding assays and aggregation time-course experiments. J.W. and C.A.H. analysed binding 164 data. L.M.N. performed the single-aggregate fluorescence imaging, super-resolution imaging 165 and analyses. J.W. and J.A.V. performed fluorescence polarisation experiments. J.A.V.
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