An expansion of the hexanucleotide (GGGGCC) repeat sequencein chromosome 9 open frame 72 (c9orf72) is the mostcommon genetic mutation in amyotrophic lateral sclerosis (ALS) andfrontotemporal dementia (FTD). The mutation leads to the productionof toxic dipeptide repeat proteins (DPRs) that induce neurodegeneration.However, the fundamental physicochemical properties of DPRs remainlargely unknown due to their limited availability. Here, we synthesizedthe c9orf72 DPRs poly-glycine-arginine (poly-GR),poly-proline-arginine (poly-PR), poly-glycine-proline (poly-GP), poly-proline-alanine(poly-PA), and poly-glycine-alanine (poly-GA) using automated fast-flowpeptide synthesis (AFPS) and achieved single-domain chemical synthesisof proteins with up to 200 amino acids. Circular dichroism spectroscopyof the synthetic DPRs revealed that proline-containing poly-PR, poly-GP,and poly-PA could adopt polyproline II-like helical secondary structures.In addition, structural analysis by size-exclusion chromatographyindicated that longer poly-GP and poly-PA might aggregate. Furthermore,cell viability assays showed that human neuroblastoma cells culturedwith poly-GR and poly-PR with longer repeat lengths resulted in reducedcell viability, while poly-GP and poly-PA did not, thereby reproducingthe cytotoxic property of endogenous DPRs. This research demonstratesthe potential of AFPS to synthesize low-complexity peptides and proteinsnecessary for studying their pathogenic mechanisms and constructingdisease models.
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