Disease-linked mutations in UBQLN2 proline-rich region promote phase separation and liquid-to-solid phase transitions.

Liquid-liquid phase separation (LLPS) underlies the assembly of biomolecular condensates such as stress granules. Stress granule persistence or disrupted stress granule dynamics is hypothesized to lead to the characteristic protein inclusions that are a hallmark of ALS (amyotrophic lateral sclerosis) and other neurological disorders. We recently demonstrated that Ubiquilin-2 (UBQLN2), an ALS-linked protein critical for maintaining protein quality control, is recruited to stress granules in cells and undergoes LLPS in vitro under physiological conditions. Mutations in the intrinsically-disordered, proline-rich (Pxx) region of UBQLN2 cause ALS and ALS/dementia and are linked to protein inclusions that form in degenerated motor neurons. The molecular mechanisms for how these Pxx mutations cause disease are unknown or poorly understood. We hypothesized that Pxx mutations disrupt UBQLN2 LLPS. Using spectrophotometric assays, light and fluorescence microscopy, we show that a subset of these mutations at positions T487, P497 or P506 significantly increase UBQLN2 LLPS propensity and/or alter material properties of UBQLN2 protein droplets in vitro. Importantly, these UBQLN2 mutants still undergo LLPS reversibly, and are all eliminated by ubiquitin binding. Biophysical characterization reveal that these single point mutations do not alter UBQLN2 structure, but likely promote UBQLN2 self-association and oligomerization, a prerequisite for LLPS. Our preliminary results suggested that increased hydrophobicity of the amino acid promotes UBQLN2 LLPS. We speculate that increased hydrophobicity promotes UBQLN2 oligomerization and self-assembly, which in turn, promotes UBQLN2 phase separation. Overall, our experiments suggest that disease-linked mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2-containing biomolecular condensates in the cell, promoting disease states.