ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner

Abstract

Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins

“…All three brain-expressed ubiquilins are linked to neurodegenerative diseases characterized by protein dyshomeostasis [1][2][3]5,28 . Recent studies highlight the propensity of UBQLN2 to form amyloid-like fibrils and liquid-like condensates 13,19,20 , but the relative aggregation behavior of these three highly homologous proteins has not been investigated before. Here we report that the three ubiquilins share the property of liquid-liquid phase transition, yet differ in condensate properties ( With respect to the shared property of liquid-liquid phase transition, UBQLN1 is most liquid-like and UBQLN4 most aggregation-prone among the three tested ubiquilins.…”

“…Like many other proteins associated with neurodegenerative diseases [13][14][15][16][17][18] , UBQLN2 spontaneously phase separates to form condensates, or liquid droplets, in which proteins are concentrated yet remain mobile 13,19,20 . Liquid-like condensates are tightly regulated by cells and can provide sites for specific cellular functions [21][22][23][24] .…”

“…At the same time, dysregulated condensate formation by various proteins leads to abnormal protein accumulation and disease 26,27 . In vitro, high salt concentrations promote UBQLN2 phase separation 19,20 . Large punctate UBQLN2 assemblies resembling liquid condensates form in cells overexpressing UBQLN2 13 .…”

“…Large punctate UBQLN2 assemblies resembling liquid condensates form in cells overexpressing UBQLN2 13 . In particular, mutations in a proline-rich domain present in UBQLN2 mediate the propensity of UBQLN2 to form less mobile liquid condensates and fibrillar aggregates 13,20 , suggesting that dynamic changes to ubiquilin condensation may underlie toxicity in mutation-driven disease.…”

Ligand Effects on Phase Separation of Multivalent Macromolecules

“…Recent studies suggest that scaffolds, which are multivalent macromolecules, can be thought of as biological instantiations of associative polymers (23,24) and modeled using the socalled stickers-and-spacers framework (4, [25][26][27][28][29]. Accordingly, the valence (number) of stickers, the sticker interaction strengths, and the solvation properties of spacers contribute as determinants of the phase behavior of multivalent protein and RNA molecules (30)(31)(32).…”