Amyloids “at the border”: deep mutagenesis and random sequence extension reveal an incomplete amyloid-forming motif in Bri2 that turns amyloidogenic upon C-terminal extension

Stop-loss mutations cause over twenty different diseases. The effects of stop-loss mutations can have multiple consequences that are, however, hard to predict. Stop-loss in ITM2B/BRI2 results in C-terminal extension of the encoded protein and, upon furin cleavage, in the production of two 34 amino acid long peptides, ADan and ABri, that accumulate as amyloids in the brains of patients affected by familial Danish and British Dementia. To systematically explore the consequences of Bri2 C-terminal extension, here, we measure amyloid formation for 676 ADan substitutions and identify the region that forms the putative amyloid core of ADan fibrils, located between positions 20 and 26, where stop-loss occurs. Moreover, we measure amyloid formation for ~18,000 random C-terminal extensions of Bri2 and find that ~32% of these sequences can nucleate amyloids. We find that the amino acid composition of these nucleating sequences varies with peptide length and that short extensions of 2 specific amino acids (Aliphatics, Aromatics and Cysteines) are sufficient to generate novel amyloid cores. Overall, our results show that the C-terminus of Bri2 contains an incomplete amyloid motif that can turn amyloidogenic upon extension. C-terminal extension with de novo formation of amyloid motifs may thus be a widespread pathogenic mechanism resulting from stop-loss, highlighting the importance of determining the impact of these mutations for other sequences across the genome.终止密码子丢失突变（stop-loss mutations）可诱发二十余种不同疾病，该类突变的效应具有多重潜在影响，却往往难以精准预测。ITM2B/BRI2基因发生的终止密码子丢失突变会导致其编码蛋白的C端延伸，经弗林蛋白酶（furin）切割后，可产生两条长度为34个氨基酸的肽段——ADan与ABri，二者会在家族性丹麦痴呆症与家族性英国痴呆症患者的大脑中以淀粉样蛋白（amyloids）的形式沉积聚集。为系统探究Bri2 C端延伸所产生的生物学效应，本研究针对676种ADan变体的淀粉样蛋白形成能力开展检测，成功定位到ADan淀粉样原纤维的推定核心区域位于第20至26位氨基酸位点，该区域恰好为终止密码子丢失突变的发生位置。此外，我们针对约18000条Bri2 C端延伸的随机序列进行淀粉样蛋白形成能力检测，发现其中约32%的序列可诱导淀粉样蛋白成核。研究表明，此类具有成核能力的序列的氨基酸组成会随肽段长度发生动态变化，仅由两类特定氨基酸（脂肪族氨基酸、芳香族氨基酸与半胱氨酸）构成的短延伸序列，即可形成全新的淀粉样蛋白核心。综上，本研究结果证实，Bri2的C端包含一个不完整的淀粉样蛋白基序，该基序可通过延伸转变为具有淀粉样蛋白形成活性的序列。由此可见，通过C端延伸从头形成淀粉样蛋白基序，或许是终止密码子丢失突变引发疾病的一种广泛存在的致病机制，这也凸显了针对基因组中其他序列的这类突变影响进行系统鉴定的重要性。