Biology subjects

Sangar, D.

Publications and source records attributed to Sangar, D..

3 recordsLinked to original sources

bioRxiv2022

Loss of the first β-strand of human prion protein generates an aggregation-competent partially "open" form

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). The pathway of PrP misfolding is still unclear, though previous data indicate the presence of a structural core in cellular PrP (PrPC), whose cooperative unfolding presents a substantial energy barrier on the path to prion formation. PrP is a GPI-anchored membrane protein, and a number of studies suggest that membrane interactions play an important role in the conversion of PrPC to its disease-associated form, including a transmembrane form of PrP in which a highly conserved region (residues 110 - 136) spans the ER membrane. Insertion of this region results in the detachment of the PrPC first {beta}-strand from the structural core. The effect of this removal on the structure, stability and self-association of the folded domain of PrPC is determined here through a biophysical characterisation of a truncated form of PrPC lacking this region. Whilst markedly destabilised, NMR chemical shifts show that the truncated protein exhibits tertiary structure characteristic of a fully folded protein and retains its native secondary structure elements, including the second strand of the PrP {beta}-sheet, but with altered conformational flexibility in the {beta}2-2 loop and first -helix. The latter is destabilised relative to the other helical regions of the protein, with markedly increased solvent exposure. This truncated form of PrP fibrilises more readily than the native form of the protein. These data suggest a stepwise mechanism, in which a destabilised "open" form of PrPC may be a key intermediate in the refolding to the fibrillar, pathogenic form of the protein.

biophysics↗

bioRxiv2022

Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed counterintuitively that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an anti-chaperone, which promotes toxic aggregation intermediates by inhibiting fibril formation.

biochemistry↗

bioRxiv2022

Direct Observation of Competing Prion Protein Fibril Populations with Distinct Structures and Kinetics

In prion diseases, fibrillar assemblies of misfolded prion protein (PrP) self-propagate by incorporating PrP monomers. Using total internal reflection and transient amyloid binding super-resolution microscopy, our study analyses elongation of single PrP fibrils to reveal polymorphic populations, featuring structural and dynamic heterogeneity similar to prion strains, which were previously hidden in ensemble measurements. PrP fibrils elongated along a preferred direction by an intermittent stop- and-go mechanism. Fibrils fell into three main populations, which each displayed distinct elongation mechanisms incorporating different monomer structures and which maintained their properties even under elongation conditions favouring a different fibril type. Elongation of RML and ME7 prion rods likewise exhibited unique kinetic features. The discovery of polymorphic fibril populations of amyloid and prions growing in competition suggests that prions may present as quasispecies of structural isomorphs and that the replication environment may tilt the balance between prion isomorphs and amyloid species. HighlightsO_LISynthetic prion fibril populations contain structurally distinct fibril types C_LIO_LIFibril types faithfully elongate by different mechanisms C_LIO_LIFibril types compete for substrate depending on environment C_LIO_LIFibril populations model quasi-species behavior of prion strains C_LI eTOCReplication of different prion strains causes distinct disease phenotypes. Sun et al. analyzed the growth of individual synthetic prion protein fibrils by super-resolution microscopy and found populations of structurally distinct fibril types, which grew in competition to each other as a quasi-species, recapitulating basic prion strain characteristics in vitro.

biophysics↗