PT - JOURNAL ARTICLE AU - Sangar, Daljit AU - Hill, Elizabeth AU - Jack, Kezia AU - Batchelor, Mark AU - Mistry, Beenaben AU - Ribes, Juan M. AU - Jackson, Graham S. AU - Mead, Simon AU - Bieschke, Jan TI - Syntaxin-6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates AID - 10.1101/2022.08.25.505283 DP - 2023 Jan 01 TA - bioRxiv PG - 2022.08.25.505283 4099 - http://biorxiv.org/content/early/2023/09/21/2022.08.25.505283.short 4100 - http://biorxiv.org/content/early/2023/09/21/2022.08.25.505283.full AB - 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.Competing Interest StatementThe authors have declared no competing interest.