Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

Wake, N.; Alcalde, J.; Jutzi, D.; Bajaj, A.; Kour, S.; Barai, M.; Weng, S.-L.; Cummings, S.; Zheng, T.; Anderson, E. N.; Wang, S.-H.; Puterbaugh, R. Z.; Bosco, D. A.; Schuster, B. S.; Mittal, J.; Pandey, U. B.; Ruepp, M.-D.; Fawzi, N. L.

doi:10.64898/2026.02.17.706410

bioRxiv · 10.64898/2026.02.17.706410

Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

Wake, N.·Alcalde, J.·Jutzi, D.·Bajaj, A.·Kour, S.·Barai, M.·Weng, S.-L.·Cummings, S.·Zheng, T.·Anderson, E. N.·Wang, S.-H.·Puterbaugh, R. Z.·Bosco, D. A.·Schuster, B. S.·Mittal, J.·Pandey, U. B.·Ruepp, M.-D.·Fawzi, N. L.

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Abstract

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting essential functions and phase separation. {beta}-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/706410v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@d15f63org.highwire.dtl.DTLVardef@1cd6221org.highwire.dtl.DTLVardef@e58126org.highwire.dtl.DTLVardef@181ec67_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYThe RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of {beta}-sheets in both function and pathological transition, we engineered {beta}-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate {beta}-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting {beta}-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTSO_LITargeted proline additions disrupt {beta}-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior C_LIO_LI{beta}-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions C_LIO_LIIn vivo models reveal attenuated toxicity of {beta}-sheet-deficient FUS in Drosophila C_LIO_LI{beta}-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology C_LI

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Wake, N., Alcalde, J., Jutzi, D., Bajaj, A., Kour, S., Barai, M., Weng, S.-L., Cummings, S., Zheng, T., Anderson, E. N., Wang, S.-H., Puterbaugh, R. Z., Bosco, D. A., Schuster, B. S., Mittal, J., Pandey, U. B., Ruepp, M.-D., Fawzi, N. L.. 2026-02-18. Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity. https://doi.org/10.64898/2026.02.17.706410

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