Deep mutagenesis reveals the distinct mutational landscape of ADan and ABri amyloid nucleation

Different forms of dementia are caused by stop-loss mutations in the ITM2B gene, also known as Bri2, which result in the expression of 34 amino acid long peptides that accumulate as amyloids in human brains. In order to gather mechanistic insights into the formation of amyloids by two of these peptides, ADan and ABri - hallmarks of Danish and British dementia respectively - we employed saturation mutagenesis combined to a massively parallel selection assay that reports on amyloid nucleation. Our results reveal that ADan aggregates into amyloids remarkably faster than both the unextended peptide Bri2 and the extended ABri sequence. The complete mutational landscape of ADan reveals asparagines and charged residues as key players in the nucleation process in addition to aliphatic residues within positions 20-25. What is more, we show that extending Bri2 with just two specific residues is enough to generate a novel amyloid core which we suggest builds the structured core of ADan fibrils. On the other hand, only a handful of mutations can boost the ability of ABri to nucleate amyloids, including a SNV replacing the Bri2 stop codon by a Cys codon. Overall, the remarkably different aggregation profiles and mutational landscapes for the two peptides suggest that different disease mechanisms underlie disease in Danish and British dementia and highlight the importance of accurately measuring the impact of stop extension mutations for these and other sequences across the genome. ### Competing Interest Statement The authors have declared no competing interest.