Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

Abstract

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…Phosphorylation introduces negative charges into proteins and changes the charge distribution and the electrostatic interactions along the polypeptide backbone, and hence also the LLPS characteristics of proteins. For example, phosphorylation regulates LLPS of nephrine (Li et al , 2012), FUS (Monahan et al , 2017), and RNA binding protein CPEB4 (Guillén‐Boixet et al , 2016). Phosphorylation is also the major type of PTMs in tau (Morris et al , 2015), and more than 80 potential phosphorylation sites can be found in the amino acid sequence of tau (Reynolds et al , 2008).…”

Protein Phase Separation: A New Phase in Cell Biology

“…The range of sequence biases associated with IDRs that mediate phase separation indicates that there may be a range of underlying driving forces. These likely include electrostatic, dipole–dipole, pi–pi, cation–pi, hydrophobic, and hydrogen bonding interactions [9,49,50,52–56] (Figure 2A,B). Indeed, mutational studies have demonstrated that phase separation of different LCDs can be prevented by interfering with a variety of residue types [9,10,14,16,57].…”

“…Work from different labs has shown that both serine and tyrosine phosphorylation can control phase separation [12,52,89,90], and the same holds true for arginine methylation [49,91] and sumoylation [80]. Importantly, the activity of the dual specificity kinase DYRK3, which partitions into SG, was shown to be necessary for SG dissolution [92], suggesting that there might be specific cellular switches controlling these processes.…”

“…Interestingly, proteins prone to phase separation, seem to be enriched in residues that are targeted by PTMs [38]. Indeed, PTMs can dramatically alter the charge or other properties of these IDRs/LCDs, hence, modifying the sequence-intrinsic driving forces to phase separate [49,52] (Figure 2C). …”

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

“…Most amazingly, this mechanism offers the potential to generate conformational ensembles composed of enormous amounts of states for both liquid droplets and fibril structures, which are also extremely sensitive to various environmental factors such as pH and cellular processes, including phosphorylation of Ser, Thr and Tyr (Murray et al 2017;Monahan et al 2017;Shorter 2017). Therefore, it appears most likely that the biological functions of the prion-like domains request not just the formation of the liquid droplets but also the conformational dynamics, as well as further assembly into diverse fibril structures.…”