Protein Structure-based FUS Mutational Subtypes Are Associated With Protein Mislocalization in Amyotrophic Lateral Sclerosis Patients.

The mislocalization of RNA-binding proteins (RBPs) from nucleus to cytoplasm and the formation of aggregates are hallmarks of neurodegeneration. Amyotrophic lateral sclerosis (ALS) disease-causing mutations in the fused in sarcoma (FUS) gene, encoding an RNA-binding protein, cluster at the C-terminal proline/tyrosine-nuclear localization signal (PY-NLS) domain, which is crucial for mediating nucleus-cytoplasm translocation by binding to Transportin-1. However, the mechanisms underlying heterogeneous protein mislocalization and age at onset (AAO) of ALS cases carrying FUS PY-NLS mutations remain unclear. Here, we screened FUS mutations in 416 ALS patients, and identified 12 patients carrying four FUS mutations at the p.R521 locus of PY-NLS domain (p.R521P, p.R521C, p.R521G, p.R521H), exhibiting highly variable AAO (20-56 years). AlphaFold-2 predicted protein structures classified FUS p.R521 mutants into alpha-helix containing (p.R521C, p.R521H) and alpha-helix disrupted (p.R521P, p.R521G) subgroups. Isothermal titration calorimetry experiment showed that the FUS alpha-helix disrupted subgroup had a reduced binding affinity with transportin-1, which is essential for mediating the nucleus-cytoplasm translocation. Furthermore, immunofluorescence in HEK-293 T and SH-SY5Y cells revealed more protein mislocalization in the FUS alpha-helix disrupted subgroup compared to the alpha-helix containing subgroup. FUS mislocalization status is also significantly associated with ALS AAO. Finally, the alpha-helix structure based FUS-ALS subgroups exhibited significantly different AAO (P = 0.036) in our cohort, but not in a Chinese cohort including published dataset. In summary, we showed highly diverse phenotypes in ALS patients with FUS R521 mutants, and implicated a link between genetic mutation related C-terminal structure with the status of FUS protein mislocalization.