ALS‐linked Mutations Modulate Ubiquilin‐2 Liquid‐Liquid Phase Separation
Authors: Lei Yongna , Dao Thuy , Hehnly Heidi , Castañeda Carlos 
Published: June 2021

Cytoplasmic protein inclusions in post‐mortem motor neurons are a pathological hallmark of amyotrophic lateral sclerosis (ALS). Inclusions are hypothesized to arise from persistent stress granules. Stress granules are stress‐induced cytosolic ribonucleoprotein assemblies that form via liquid‐liquid phase separation (LLPS). They can dissipate when stress is removed; however, disease‐linked mutations in their protein components can disrupt stress granule dynamics and make them persist in cells. Persistent stress granules are hypothesized to accumulate and mature into cytoplasmic inclusions. Stress granule maintenance is regulated by the protein quality control (PQC) system. One of the PQC members is an ALS‐linked protein called ubiquilin‐2 (UBQLN2), which acts as a proteasomal shuttle factor and mediates protein degradation through the ubiquitin‐proteasome system and autophagy. Importantly, both wild‐type (WT) and mutant UBQLN2 have been found in inclusions in ALS patients. We previously demonstrated that UBQLN2 is recruited to stress granules in cells under different stress conditions and undergoes reversible LLPS in vitro under physiological conditions. UBQLN2 LLPS, a process in which proteins demix into liquid‐like droplets in solution, is driven by weak, multivalent interactions and is eliminated upon ubiquitin (Ub) binding. Many ALS‐linked mutations in UBQLN2 cluster to the proline‐rich Pxx region, which is unique to UBQLN2 along the other ubiquilin paralogs in humans. We hypothesized that the ALS‐linked mutations perturb UBQLN2 LLPS, alter droplet morphology, and that these mutants might respond to Ub binding differently from wild‐type (WT) UBQLN2. To probe the effects of ALS‐linked mutations on UBQLN2 LLPS, we used turbidity spectrophotometric assays to monitor UBQLN2 droplet formation and differential interference contrast microscopy to characterize droplet morphology. We used UBQLN2 450–624 (450C), the minimum length construct that undergoes LLPS, as a control, and examined proteins harboring 11 individual point mutations in the 450C background, T487I, A488T, P497H, P497L, P497S, P500S, P506A, P506S, P506T, P509S, and P525S. We found that a subset of ALS‐linked mutations (P506S, P506T, P506A, T487I, P497L, P497H, and P497S) significantly decreases the lower critical solution temperature (LCST) and thereby promotes UBQLN2 LLPS at lower temperatures. These mutations also promote LLPS over a larger temperature range. Five mutants within this subset (P506A, T487I, P497L, P497H, and P497S) form amorphous, gel‐like droplets or even aggregates. Ub clears WT and mutant UBQLN2 droplets and aggregates on a similar timescale.Support or Funding Information1. ALS Association grants 17‐IIP‐369 and 18‐IIP‐400 2. Syracuse University Meredith Scholar Award 3. Syracuse University Renée Crown University Honors Program Lynne Parker Award 4. Syracuse University Biology DepartmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.